Encyclopedia of Physical Science and Technology, 3e, Biotechnology

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Encyclopedia of Physical Science and Technology

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Table of Contents (Subject Area: Biotechnology)

Article Biomass Utilization, Limits of Biomass, Bioengineering of Biomaterials, Synthetic Synthesis, Fabrication, and Applications Biomineralization and Biomimetic Materials Bioreactors Fiber-Optic Chemical Sensors

Authors

Pages in the Encyclopedia

David Pimentel

Pages 159-171

Bruce E. Dale

Pages 141-157

Carole C. Perry

Pages 173-191

Paul D. Calvert

Pages 193-205

Yusuf Chisti and Murray Moo-Young David R. Walt, Israel Biran and Tarun K. Mandal Michael Butler

Pages 247-271

Hybridomas, Genetic Engineering of Ferenc A. Jolesz Image-Guided Surgery Bryan Griffiths and Mammalian Cell Florian Wurm Culture Metabolic Engineering Jens Nielsen Jerome S. Schultz Microanalytical Assays Abraham Katzir Optical Fiber Techniques for Medical Applications Giancarlo Santus and Pharmaceuticals, Controlled Release of Richard W. Baker Michael F. Flessner Pharmacokinetics Laure G. Berruex and Separation and Ruth Freitag Purification of Biochemicals François Berthiaume Tissue Engineering and Martin L. Yarmush Toxicology in Forensic Olaf H. Drummer Science

Pages 803-829

Pages 427-443 Pages 583-594 Pages 31-47 Pages 391-406 Pages 679-694 Pages 315-333

Pages 791-803 Pages 805-820 Pages 651-673

Pages 817-842 Pages 905-911

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Biomass Utilization, Limits of David Pimentel Cornell University

I. II. III. IV. V. VI.

Biomass Resources Conversion of Biomass Resources Biogas Biomass and the Environment Social and Economic Impacts Conclusion

GLOSSARY Biodiversity All species of plants, animals, and microbes in one ecosystem or world. Biogas A mixture of methane and carbon dioxide produced by the bacterial decomposition of organic wastes and used as a fuel. Biomass Amount of living matter, including plants, animals, and microbes. Energy Energy is the capacity to do work and includes heat, light, chemical, acoustical, mechanical, and electrical. Erosion The slow breakdown of rock or the movement and transport of soil from one location to another. Soil erosion in crop and livestock production is considered serious worldwide. Ethanol Also called ethyl alcohol. A colorless volatile flammable liquid with the chemical formula C2 H5 OH that is the intoxicating agent in liquors and is also used as a solvent. Methanol Also called methyl alcohol. A light volatile flammable liquid with the chemical formula CH3 OH that is used especially as a solvent, antifreeze, or

denaturant for ethyl alcohol and in the synthesis of other chemicals. Pollution The introduction of foreign, usually manmade, products or waste into the environment. Pyrolysis Chemical change brought about by the action of heat. Subsidy A grant or gift of money.

THE INTERDEPENDENCY of plants, animals, and microbes in natural ecosystems has survived well for billions of years even though they only captured 0.1% of the sun’s energy. All the solar energy captured by vegetation and converted into plant biomass provides basic resources for all life, including humans. Approximately 50% of the world’s biomass is used by humans for food plus lumber and pulp and medicines, as well as support for all other animals and microbes in the natural ecosystem. In addition some biomass is converted into fuel. Serious shortages of biomass for human use and maintaining the biodiversity in natural ecosystems now exist throughout the world. Consider that more than 3 billion humans are now malnourished, short of food, and various

159

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160 essential nutrients. This is the largest number and proportion of malnourished humans ever recorded in history. Meanwhile, based on current rates of increase, the world population is projected to double to more than 12 billion in approximately 50 years. With a population growth of this magnitude, the numbers of malnourished could reach 5 billion within a few decades. The need for biomass will continue to escalate. Associated with increasing human numbers are diverse environmental problems, including deforestation, urbanization, industrialization, and chemical pollution. All these changes negatively impact on biomass production that is vital to human life and biodiversity. However, at present and in the foreseeable future the needs of the rapidly growing human population will stress biomass supplies. In our need to supply food and forest products for humans from biomass, intense competition between human needs for food and the conversion of biomass into an energy resource is expected to intensify in the coming decades. Furthermore, human intrusion throughout the natural environment is causing a serious loss of biodiversity with as many as 150 species being lost per day. The present rate of extinction of some groups of organisms is 1000–10,000 times faster than that in natural systems. Ecosystem and species diversity are the vital reservoir of genetic material for the successful development of agriculture, forestry, pharmaceutical products, and biosphere services in the future. The limits of biomass energy utilization and how this relates to food production and natural biodiversity and environmental quality are discussed in this article.

I. BIOMASS RESOURCES The amount of biomass available is limited because plants on average capture only about 0.1% of the solar energy reaching the earth. Temperature, water availability, soil nutrients, and feeding pressure of herbivores all limit biomass production in any given region. Under optimal growing conditions, natural and agricultural vegetation and produce about 12 million kilocalories per hectare per year (about 3 t/ha dry biomass). A. World Biomass The productive ecosystems in the world total an estimated 50 billion hectare, excluding the icecaps. Marine ecosystems occupy approximately 36.5 billion hectare while the terrestrial ecosystems occupy approximately 13.5 billion hectare. Gross primary productivity for the marine ecosystem is estimated to be about 1 t/ha/yr, making the to-

Biomass Utilization, Limits of

tal biomass production about 36.5 billion metric tons or 145 × 1015 kcal/yr. In contrast, the terrestrial ecosystem produces about 3 t/ha/yr, making the total biomass about 40.5 billion tons or 162 × 1015 kcal/yr. The total biomass produced is approximately 77 billion tons or about 12.8 t per person per year. The 40.5 billion tons of biomass produced in the terrestrial ecosystem provides an estimated 6.8 t/yr per person. Given that humans harvest about 50% of the world’s terrestrial biomass, each person is utilizing 3.4 t/yr. This 3.4 t/yr includes all of agriculture, including livestock production and forestry. The remaining 3.4 t/yr per person supplies the other 10 million species of natural biota their energy and nutrient needs. Currently, approximately 50% of the world’s biomass (approximately 600 quads worldwide) is being used by humans for food, construction, and fuel. This major utilization of biomass, habitat destruction associated with the rapid increase in the world population, and environmental pollution from about 100,000 chemicals used by humans is causing the serious loss of biodiversity worldwide. With each passing day an estimated 150 species are being eliminated because of increasing human numbers and associated human activities, including deforestation, soil and water pollution, pesticide use, urbanization, and industrialization. B. United States Biomass In the North American temperate region, the solar energy reaching a hectare of land per year is 14 billion kilocalories. However, plants do not grow during the winter there. Most plant growth occurs during 4 months in the summer when about 7 billion kilocalories reach a hectare. In addition to low temperatures, plant growth is limited by shortages of water, nitrogen, phosphorus, potassium, and other nutrients, plus the feeding pressure of herbivores and disease organisms. At most, during a warm moist day in July a plant, like corn, under very favorable conditions, might capture only 5% of the sunlight energy reaching the plants. Under natural and agricultural conditions for the total year, vegetation produces approximately 12 million kilocalories per hectare per year or about 3 t/ha dry biomass. Total annual biomass produced in the United States is an estimated 2.6 billion tons (Table I). This is slightly more than 6% of all the terrestrial biomass produced in the world. Based on the United States. land area of 917 million hectares, this is the equivalent of 2.9 t/ha/yr and is similar to the world average of 3 t/ha/yr for all the terrestrial ecosystems of the world. The total energy captured by all the United States plant biomass each year is approximately 11.8 × 1015 kcal (Table I). With the United States currently consuming 87 quads (21.8 × 1015 kcal)

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161

Biomass Utilization, Limits of TABLE I Annual Biomass Production in the United States

Cropland and crops Pasture and forage Forests Other Total area Total biomass Total energy (1015 /kcal) Biomass production (t/ha)

Land area (106 /ha)

Biomass production (106 /t)

192 300 290 135 917 —

1,083 900 580 68 — 2,631

11.8 2.9

[From Pimentel, D., and Kounang, N. (1998), Ecosystems 1, 416–426.]

of fossil energy each year, this means that it is consuming 85% more fossil energy than the total energy captured by all its plant biomass each year. C. United States Agricultural and Forest Products and Biofuels Including crops and forages from pastures, the United States harvests approximately 1307 million tons of biomass per year in agricultural products and approximately 100 million tons of biomass per year as forest products (Table II). Together the energy value of harvested agricultural and forest products total 6352 1012 kcal/yr (Table II). These data suggest that the United States is harvesting in the form of agricultural and forest products, 54% of the total energy captured each year by the United States biomass annually (Tables I and II). This total does not include the biomass harvested now and used as biofuel.

II. CONVERSION OF BIOMASS RESOURCES In addition to using biomass directly as food, fiber, lumber, and pulp, biomass is utilized as a fuel. The total biofuel utilized in the United States is slightly more than 3 quads (800 × 1012 kcal) per year. If the biofuel energy is added to that harvested as agricultural and forest products, then the total biomass energy harvested from the United States terrestrial ecosystem is 7332 × 1012 kcal/yr. This is equivalent to 62% of the total biomass energy produced in the United States each year. Harvesting this 62% is having a negative impact on biodiversity in the nation. A. Direct Heating Heat production is the most common conversion system for using biomass resources. Heat from wood and other biomass resources is utilized for cooking food, heating homes, and producing steam for industry.

Each year, worldwide, an estimated 5300 million dry tons of biomass are burned directly as a fuel, providing about 88 quads of energy. Rural poor in developing countries obtain up to 90% of their energy needs by burning biomass. In developing countries, about 2 billion tons of fuelwood, 1.3 billion tons of crop residues, plus nearly 1 billion tons of dung are burned each year. Although some deforestation results from the use of fuelwood, the most significant environmental impacts result from burning crop residues and dung. When crop residues and dung are removed from the land and used as a fuel this leaves the cropland without vegetative protection and exposed to wind and water erosion. Erosion destroys the productivity of cropland, by robbing the soil of nutrients, essential water, soil organic matter, and adequate rooting depth. Cooking requires relatively large amounts of fuel and is essential for preventing disease, improving nutrition, and increasing the palatability of many foods. The transfer of heat from the woodfire in a stove to the food product is about 33% efficient, while over an open fire, the heat transfer to the food is only about 10% efficient. Under usual cooking conditions, from 2 to 3 kcal are required to cook 1 kcal of food. TABLE II Total Annual Amount of Solar Energy Harvested in the Form of Agricultural and Forest Biomass in the U.S. Tons (106 ) Corn Wheat Rice Soybeans Sorghum Potatoes Vegetables Fruits Nuts Oil seeds Sugarcane Sugar beets Pulses Oats Rye Barley Total Pasture forage Forest products Totals Total per capita (tons) Total per capita (106 /kcal)

Energy (1012 kcal)

194 71 6 51 22 16 6 5 0.8 9 2.5 2 1 7 1 13 407.3 900 100 1,407

873 320 27 230 99 72 27 23 4 41 20 27 5 32 5 59 1,853 4,050 450 6,352 5.2 23.3

[From Pimentel, D., and Kounang, N. (1998), Ecosystems 1, 416– 426.]

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162 In a developing country an average, 600–700 kg/yr of dry biomass per person is used for cooking. For example, the use of fuelwood for cooking and heating in Nepal is about 846 kg/yr of biomass per person. Other investigators report that from 912 to 1200 kg/yr of biomass per person is used for both cooking and heating. In some developing countries, fuelwood for cooking and heating may cost almost as much as the food, making it necessary to use crop residues and dung. A significant amount of wood is converted into charcoal for cooking and heating. Similar to wood fires for cooking, open charcoal fires are only about 10% efficient in transferring heat energy to food. However, charcoal has some advantages over wood. First, it is lightweight and easy to transport. One kilogram of charcoal contains about 7100 kcal of potential energy in contrast to a kilogram of wood that has about 4000 kcal. Charcoal burns more uniformly and with less smoke than wood. However, charcoal production is an energy-intensive process. Although charcoal has a high energy content, from 20,300 to 28,400 kcal of hardwood must be processed to obtain the 7100 kcal of charcoal. Considering this low conversion efficiency ranging from 25 to 35%, charcoal heating for cooking has an overall energy transfer efficiency to food of only 2.5–3.5%. Further, the use of charcoal uses more forest biomass than directly burning the wood. Using fuelwood for the production of steam in a boiler under relatively optimal conditions is 55–60% efficient, that is, burning 4000 kcal of air-dried wood provides from 2200 to 2400 kcal of steam in the boiler. More often the efficiency is less than 55–60%. Steam production is used to produce electricity and producing a salable product, such as steam, for industrial use. Collecting biomass for fuel requires a substantial amount of time and human effort. For example, in Indonesia, India, Ghana, Mozambique, and Peru families spend from 1.5 to 5 hrs each day collecting biomass to use as a fuel. Estimates are that more than half of the people who depend on fuelwood have inadequate supplies. In some countries, such as Brazil, where forest areas are at present fairly abundant, the rural poor burn mostly wood and charcoal. However, in many developing countries crop residues account for most of the biomass fuel, e.g., 55% in China, 77% in Egypt, and 90% in Bangladesh. Estimates are that the poor in these countries spend 15–25% of their income for biomass fuel. B. Health Effects Environmentally, burning biomass is more polluting than using natural gas, but less polluting than coal. Biomass

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combustion releases more than 200 different chemical pollutants into the atmosphere. The pollutants include, up to 14 carcinogens, 4 cocarcinogens, and 6 toxins that damage cilia, plus additional mucus-coagulating agents. Wood smoke contains pollutants known to cause bronchitis, emphysema, cancer, and other serious illnesses. Globally, but especially in developing nations where people cook with fuelwood over open fires, approximately 4 billion humans suffer continuous exposure to smoke. This smoke which contains large quantities of particulate matter and more than 200 chemicals, including several carcinogens, results in pollution levels that are considerably above those acceptable by the World Health Organization (WHO). Worldwide fuelwood smoke is estimated to cause the death of 4 million children each year worldwide. In India, where people cook with fuelwood and dung, particulate concentrations in houses are reported to range from 8300 to 15,000 µg/m3 , greatly exceeding the 75 µg/m3 maximum standard for indoor particulate matter in the United States. Because of the release of pollutants, some communities in developed areas, such as Aspen, CO, have banned wood burning for heating homes. When biomass is burned continuously in a confined space for heating, its pollutants accumulate and can become a serious health threat. C. Ethanol Production Numerous studies have concluded that ethanol production does not enhance energy security, is not a renewable energy source, is not an economical fuel, and does not insure clean air. Further, its production uses land suitable for crop production and causes environmental degradation. The conversion of corn and other food/feed crops into ethanol by fermentation is a well-known and established technology. The ethanol yield from a large plant is about 9.5 l (2.5 gal) from a bushel of corn of 24.5 kg (2.6 kg/l of ethanol). Thus, a hectare of corn yielding 7965 kg/ha could be converted into about 3063 l of ethanol. The production of corn in the United States requires a significant energy and dollar investment (Table III). For example, to produce 7965 kg/ha of corn using conventional production technology requires the expenditure of about 10.4 million kcal (about 10,000 l of oil equivalents) (Table III), costing about $857.17 for the 7965 kg or approximately 10.8c/ /kg of corn produced. Thus, for a liter of ethanol, the corn feedstock alone costs 28c/ . The fossil energy input to produce the 7965 kg/ha corn feedstock is 10.4 million kilocalories or 3408 kcal/l of ethanol (Table III). Although only 16% of United States corn production is currently irrigated, it is included in the analysis, because irrigated corn production is energy costly. For the 150 mm of irrigation water applied and

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Biomass Utilization, Limits of TABLE III Energy Inputs and Costs of Corn Production per Hectare in the United States Inputs

Quantity

Labor Machinery Diesel Gasoline Nitrogen Phosphorus Potassium Lime Seeds Herbicides Insecticides Irrigation Electricity Transportation Total Corn yield = 7,965 kg

11.4 hr 55 kg 42.2 L 32.4 L 144.6 kg 62.8 kg 54.9 kg 699 kg 21 kg 3.2 kg 0.92 kg 150 mm 13.2 kg 151 kg

kcal × 1000

Costs

561 $100.00 1,018 103.21 481 8.87 328 9.40 2,668 89.65 260 34.54 179 17.02 220 139.80 520 74.81 320 64.00 92 18.40 3,072 150.00 34 2.38 125 45.30 10,439 $857.17 27,758 kcal output/kcal input = 1 : 2.66

From Pimentel, D., Doughty, R., Carothers, C., Lamberson, S., Bora, N., and Lee, K. J. Agr. Environ. Ethics (in press).

pumped from only 30.5 m (100 feet), the average energy input is 3.1 million kilocalories/hectare (Table III). When investigators ignore some of the energy inputs in biomass production and processing they reach an incomplete and deficient analysis for ethanol production. In a recent USDA report, no energy inputs were listed for machinery, irrigation, or for transportation. All of these are major energy input costs in United States corn production (Table III). Another way of reducing the energy inputs for ethanol production is to arbitrarily select lower production costs for the inputs. For instance, Shapouri et al. list the cost of a kilogram of nitrogen production at 12,000 kcal/kg, considerably lower than Food and Agricultural Organization of the UN (FAO), which list the cost of nitrogen production at 18,590 kcal/kg. Using the lower figure reduces the energy inputs in corn production by about 50%. Other workers have used a similar approach to that of Shapouri et al. The average costs in terms of energy and dollars for a large (240 to 280 million liters per year), modern ethanol plant are listed in Table IV. Note the largest energy inputs are for corn production and for the fuel energy used in the fermentation/distillation process. The total energy input to produce 1000 l of ethanol is 8.7 million kilocalories (Table IV). However, 1000 l of ethanol has an energy value of only 5.1 million kilocalories. Thus, there is a net energy loss of 3.6 million kilocalories per 1000 l of ethanol produced. Put another way, about 70% more energy is re-

quired to produce 1000 l of ethanol than the energy that actually is in the ethanol (Table IV). In the distillation process, large amounts of fossil energy are required to remove the 8% ethanol out of the 92% water. For example, to obtain 1000 l of pure ethanol with an 8% ethanol concentration out of 92% water, then this ethanol must come from the 12,500 l of ethanol/water mixture. A total of 124 l of water must be eliminated per liter of ethanol produced. Although ethanol boils at about 78◦ C, in contrast to water at 100◦ C, the ethanol is not extracted from the water in one distillation process. Instead, about 3 distillations are required to obtain the 95% pure ethanol that can be mixed with gasoline. To be mixed with gasoline, the 95% ethanol must be further processed with more energy inputs to achieve 99.8% pure ethanol. The three distillations account for the large quantities of fossil energy that are required in the fermentation/distillation process. Note, in this analysis all the added energy inputs for fermentation/distillation process are included, not just the fuel for the distillation process itself. This contrasts with Shapouri et al. who, in 1995, give only one figure for the fermentation/distillation process and do not state what the 3.4 million kilocalories represents in their analysis for producing 1000 l of ethanol. Careful and detailed analyses and full accountings are needed to ascertain the practicality of ethanol production as a viable energy alternative. About 61% of the cost of producing ethanol (46c/ per liter) in such a large-production plant is for the corn substrate itself (28c/ /l) (Table IV). The next largest input is for coal to fuel the fermentation/distillation process, but this was only 4c/ (Table IV). These ethanol production costs include a small charge for pollution control (6c/ per liter), which is probably a low estimate. In smaller plants with an annual production of 150,000 l/yr, the cost per liter increases to as much as 66c/ per liter. Overall, the per liter TABLE IV Inputs per 1000 l of Ethanol Produced from Corn Inputs

Kilograms

Kilocalories (1000)

Dollars

Corn Transport of corn Water Stainless steel Steel Cement Coal Pollution control costs Total

2,600 2,600 160,000 6 12 32 660 —

3,408 312 90 89 139 60 4,617 — 8,715

$280 32 20 10 10 10 40 60 $462

From Pimentel, D., Warneke, A. F., Teel, W. S., Schwab, K. A., Simcox, N. J., Ebert, D. M., Baenisch, K. D., and Aaron, M. R., (1988). Adv. Food. Res. 32, 185–238.

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164 price for ethanol does not compare favorably with that for the production of gasoline fuels which presently is about 25c/ per liter. Based on current ethanol production technology and recent oil prices, ethanol still costs substantially more to produce in dollars than it is worth on the market. Clearly, without the approximately $1 billion subsidy, United States ethanol production would be reduced or cease, confirming the fact that basically ethanol production is uneconomical. Federal subsidies average 16c/ per liter and state subsidies average 5c/ per liter. Because of the relatively low energy content of ethanol, 1.5 l of ethanol is the energy equivalent of 1 l of gasoline. This means that the cost of subsidized ethanol is 68c/ per liter. The current cost of producing gasoline is about 25c/ per liter. At present, federal and state subsidies for ethanol production total about $1 billion per year and are mainly paid to large corporations (calculated from the above data). The costs to the consumer are greater than the $1 billion per year used to subsidize ethanol production because of increased corn prices. The resulting higher corn prices translate into higher meat, milk, and egg prices because currently about 70% of the corn grain is fed to United States livestock. Doubling ethanol production can be expected to inflate corn prices perhaps as much as 1%. Therefore, in addition to paying tax dollars for ethanol subsidies, consumers would be paying significantly higher food prices in the market place. It should be noted that the USDA is proposing to increase the subsidies to the large corporations by about $400 million per year. Currently about 3.8 billion liters of ethanol are being produced in the United States each year. This amount of ethanol provides only about 1% of the fuel utilized by United States automobiles. To produce the 3.8 billion liters of ethanol we must use about 1.3 million hectares of land. If we produced 10% of United States fuel the land requirement would be 13 million hectares. Moreover not all the 3.8 billion liters would be available to use, because a lot would be needed to sow, fertilize, and harvest 13 million hectares. Clearly, corn is not a renewable resource for ethanol energy production. The energy and dollar costs of producing ethanol can be offset in part by the by-products produced, especially the dry distillers grains (DDG) made from dry-milling that can be fed primarily to cattle. Wet-milling ethanol plants produce such by-products as corn gluten meal, gluten feed, and oil. Sales of the by-products help offset the energy and economic costs of ethanol production. For example, use of by-products can offset the ethanol production costs by 8–24% (Table IV). The resulting energy output/input comparison, however, remains negative (Table IV). The sales of the by-products that range from13 to 16c/ per liter do not make ethanol competitive with gasoline.

Biomass Utilization, Limits of

Furthermore, some of the economic and energy contributions of the by-products are negated by the environmental pollution costs associated with ethanol production. These are estimated to be about 6c/ per liter (Table IV). In United States corn production, soil erodes about 12 times faster than it can be reformed. In irrigated corn acreage, ground water is being mined 25% faster than its natural recharge rate. This suggests that the environmental system in which corn is being produced is being rapidly degraded. Further, it substantiates the finding that the United States corn production system is not sustainable for the future, unless major changes are made in the cultivation of this major food/feed crop. Corn should not be considered a renewable resource for ethanol energy production. When considering the advisability of producing ethanol for automobiles, the amount of cropland required to grow corn to fuel each automobile should be understood. To clarify this, the amount of cropland needed to fuel one automobile with ethanol was calculated. An average United States automobile travels about 16,000 km/yr and uses about 1900 l/yr of gasoline. Although 8000 kg/ha of corn will yield about 3100 l of ethanol, it has an energy equivalent of only 1952 l because ethanol has a much lower kilocalories content than gasoline. However, even assuming zero or no energy charge for the fermentation and distillation process and charging only for the energy required to produce corn (Table III), the net fuel energy yield from 1 ha of corn is 433 l. Thus, to provide 1900 l per car, about 4.4 ha of corn must be grown to fuel one car with ethanol for one year. In comparison, only 0.6 ha of cropland is currently used to feed each American. Therefore, more than seven times more cropland would be required to fuel one automobile than is required to feed one American. Assuming a net production of 433 l of fuel per corn hectare and if all automobiles in the United States were fueled with ethanol, then a total of approximately 900 million hectares of cropland land would be required to provide the corn feedstock for production. This amount of cropland would equal nearly the total land area of the United States. Brazil had been a large producer of ethanol, but has abandoned subsidizing it. Without the subsidy, economic ethanol production is impossible.

III. BIOGAS Biomass material that contains large quantities of water can be effectively converted into usable energy using naturally occurring microbes in an anaerobic digestion system. These systems use feedstocks, like dung and certain plants such as water hyacinth, although production and

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harvesting costs of the latter are generally greater than for dung. The processing facility can be relatively simple and be constructed for about $700. A large facility capable of processing the dung from 320 cows might cost about $150,000. The basic principles for both systems are similar. Manure from a dairy farm or small cattle operation is loaded or pumped into a sealed, corrosion-resistant digestion tank where it is held from 14 to 28 days at temperatures from 30 to 38◦ C. In some digestion systems, the manure in the tank is constantly stirred to speed the digestion process and assure even heating. During this period, the mesophilic bacteria break down volatile solids (VS) in the manure and convert them into methane gas (65%) and carbon dioxide (35%). Small amounts of hydrogen sulfide may also be produced. This gas is drawn off through pipes and either burned directly, similar to natural gas, or scrubbed to clean away the hydrogen sulfide and used to generate electricity. The energy output/input is listed in Table V. The amount of biogas produced in this system is determined by the temperature of the system, the VS content of the feedstock, and the efficiency of converting it into

TABLE V Energy Inputs Using Anaerobic Digestion for Biogas Production from 100 t wet (13 t dry) using Cattle Manure (Pimentel et al., 1988)a,b

Inputs Labor hours Electricity Cement foundation (30-year life) Steel (gas collector and other equipment with 30-year life) Pumps and motors Truck/tractor for transport (10-year life) Fuel for transport (10-km radius) Total inputs Total biogas output

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Quantity

kcal (1,000)

20 hr 2,234 kWh 0.9 kg 35 kg

— 5,822 2 725

0.5 kg 10 kg 34 l

1 200 340 7,090 10,200

a The retention time in the digester is 20 days. The unit has the capacity to process 1,825 t (wet) per year. Note: the yield in biogas from 100 t is estimated at 10.2 million kilocalories. Thus, the net yield is 3.1 million kilocalories. The energy for heating the digester is cogenerated from the cooling system of the electric generator. b It is assumed that anaerobic digestion of the manure takes place at 35◦ C with a solids retention time of 20 days. The temperature of the fresh manure is 18◦ C, and the average ambient temperature is 13◦ C. The manure is assumed to have the following characteristics: production per cow per day, 23.6 kg total; solids, 3.36 kg; and biological oxygen demand (BOD), 0.68 kg. The digester is assumed to transform 83% of the biodegradable material into gas. The biogas produced is 65% methane, and its heat of combustion is 5720 kcal/m3 at standard conditions.

biogas. This efficiency varies from 18 to 95%. Dairy cows produce 85 kg daily of manure for each 1000 kg of live weight. The total solids in this manure average 10.6 kg, and of these, 8.6 kg are VS. Theoretically, a 100% efficient digester could produce 625 l of biogas for every kilogram of VS in the system. The digester utilized for the data presented in Table V was 28.3% efficient. It produces 177 l of biogas per kilogram of VS added or 1520 l of biogas per 1000 kg live weight of cattle daily. Note, if the total heat value of the manure was used in calculating efficiency, then the percentage efficiency would be only 5%. Biogas has an energy content of about 5720 kcal/m3 , compared to 8380 kcal/m3 for pure methane gas, because carbon dioxide is present in the biogas. Energy costs and energy outputs for processing 100 t of manure (wet), with a 7.1 million kilocalories energy input, results in a total of 10.2 million kilocalories produced for a net energy yield of 3.1 million kilocalories (Table V). Much of the energy input or cost comes from the production of electricity to run the pumps and stirring system used to reduce the retention time in the digester. The volume of the digester is determined by the amount of manure produced by the animals during the retention time. In this example, with a retention time of 14 days, it would be slightly over 75 m3 . It is assumed that the electricity is generated from the biogas and that the electrical conversion efficiency of the entire operation is 33%. The energy needed to heat the digester is cogenerated by the electric generator via the use of the generator’s cooling system as the heat source. The net energy produced by the digester can either be used to generate electricity for the farm or be used as heat source for other on-farm activities. Although material costs are lowered if there is no generator or stirring mechanism on the digester, the size of the digester must be increased because of the increased retention time needed to complete the process. Also, some of the biogas will have to be used to heat the digester, perhaps an additional 610,000 kcal for every 100 wet tons of manure digested. The critical heat requirements are calculated by including the heat losses to the surroundings, the heat associated with the feed and effluents, and the heat released by the biological reaction. In the tropics, the overall efficiency of the biogas systems is enhanced because there is no need to heat the system to keep the temperature in the 30–38◦ C range. Dairy cattle are not the only source of manure for biogas systems. They are used as a model since dairy animals are more likely to be located in a centralized system, making the collecting and adding the manure to a digestion system less time consuming and energy intensive than for range-fed steers, or even for draft animals. Efficiencies of conversion vary not only from system to system, but also the sources of manure. Swine and beef cattle manure

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166 appears to yield more gas per kilogram of VS than dairy cattle manure. Poultry manure is also used, but sand and other forms of heavy grit in this dung cause pump maintenance problems and require more frequent cleaning of the digester. Manure processed in the digester retains its fertilizer value and has the advantage of less odor. Therefore, it can be spread on fields and may be easier to pump if the initial pumping system used a cutter pump to break up stray bits of straw or long undigested fibers. Biogas systems have the advantage of being able to adjust in size according to the scale of the operation. The pollution problem associated with manure in a centralized dairy production system is the same whether or not it goes through a biogas generator. In developing countries, such as India, the situation is different. There, a substantial percentage of the manure as dried dung is burned directly as fuel. Although burning utilizes a significantly higher percentage of the total energy in the manure, it results in a complete loss of nitrogen and loss of substantial amounts of the other valuable nutrients. Whether or not biogas is a useful energy alternative in India and other similar countries is highly problematic in spite of the higher overall energy efficiency of the conversion system. If it is not desirable to produce electricity from the biogas, the energy data listed in Table V will change considerably. For instance, less energy will be lost in the conversion to electricity if all the energy is used directly for heating. However, compressing biogas for use in tractors involves the input of significant amounts of additional energy for “scrubbing” the biogas to remove hydrogen sulfide and water. A. Biogas for Smallholders The economics of biogas production in a rural area of a developing nation, like Kenya or India, illustrates that costs and benefits are complex and results mixed. The capital costs of constructing a simple biogas digester with a capacity to process 8 t (wet) of manure per 20-day retention time, or 400 kg/day, are estimated to be between $2000 and $2500 (Table VI). Such a unit would have usable life of 30 years, so the capital costs are only $80 per year. If rural workers construct the biogas generator themselves, material costs might range from $300 to $700. At $400 for materials, without any charge for labor, the investment would be only $14 per year with the costs spread out over the life of the digester. A digester this size in India, where cows weigh an average of between 225 to 330 kg each, would require access to manure from about 20 cows. This system would produce

Biomass Utilization, Limits of TABLE VI Energy Inputs for Anaerobic Digester in the Tropics for Biogas Production using 8 t (1 t dry) of Cow Manure (Pimentel et al., 1988)a

Inputs Cement foundation (30-year life) Steel (30-year life) Total inputs Total biogas output Net return per 1 t dry manure

Quantity (kg)

kcal

0.07 0.33

140 7,000 7,140 820,000 812,840

a The retention time is 20 days without a means of storing the biogas. The gas is used as delivered. The digestion takes place at 35◦ C. The temperature of the fresh manure is assumed to be 21◦ C, and the average ambient temperature is 21◦ C. The efficiency of the digester is 25%. The biogas produced is 65% methane and its heat of combustion is 5720 kcal/m3 .

an estimated 2277 m3 of biogas per year at a conversion efficiency of 25% (Table VI). The energy value of this gas totals 13.0 million kcal. Assuming $8.38 per 1 million kcal, the economic value of this much energy is $109 per year. Then if no charge is made for labor and dung and the capital cost is assumed to be only $14 per year, the net return is $95 per year. These costs are not equally applicable to Kenya where the energy replacement of biogas in terms of woodfuel saved is appropriate. Using an average of 4000 kcal/kg of woodfuel, this amount of biogas would replace 3 t of wood and since biogas is generally more efficient than wood when used for cooking, the total amount of wood replaced might be double. Although the labor requirement for the described biogas generator is only 5–10 min/day, the labor input for collecting and transporting biomass for the generator may be significant. If the source for the 400 kg of manure required for the digester was, on average, 3 km from the digester, it would take 2 laborers working an 8-hr day to collect manure, feed it into the digester, and return the manure to cropland where it could be utilized as fertilizer. On a per hour basis, the laborers would have to work for 3c/ per hour for the biogas digester to have costs equal to the amount of gas produced. In some situations, especially in densely populated parts of a country, the amount of transport required will be too costly. Although the profitability of small-scale biogas production may be low even without the charge of labor, biogas digesters have significant advantages in rural areas. The biomass can be processed and fuel energy obtained without losing the valuable nutrients (N, P, and K) present in the manure. Nitrogen and phosphorus are major limiting nutrients in tropical agriculture and these are returned to the cropland. The only loss that the processed manure

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has undergone is the breakdown of the fibrous material it contains, making it a less effective agent for the control of soil erosion. In contrast, when biomass is directly burned as a fuel, both nitrogen and other nutrients are lost to the atmosphere. The nitrogen in the biogas slurry (for the 146 t/yr amounts) would amount to about 3.7 t/yr. This has an energy value of 77 million kcal and market value of $2293. Then if the nitrogen value and the gas value combined, the return for such a system is approximately $2388. The nitrogen fertilizer value of the processed manure makes it worthwhile as a biogas source rather than burning it as a primary fuel cakes. Based on this, each laborer would receive about 60c/ per hour for his work. The total amount of manure produced annually in the United States is about one billion tons. It would be an achievement to manage to process even half of this in biodigesters. Due to low net yield of energy, as described, even 500 million t of manure, with gas produced at 28% efficiency, would provide energy for a population of 270 million Americans of 0.0076 kW per person per year. This represents only 0.0008% of present net energy use. B. Gasification Biomass wood with less than 50% moisture can be heated in the presence of air and gasified. The gas produced can be used to run internal combustion engines and also used as a gas fuel and for other purposes. When used in the internal combustion engine, the gas must be cleaned thoroughly as the several chemical contaminates it contains corrode engines and reduce its efficiency. A kilogram of air-dried biomass will produce approximately 2000 kcal of clean gas which can generate about 0.8 kWh of net power electricity. The low heating value of the gas-air mixture in a gasoline engine results in derating the engine by 30–40%. This problem can be overcome by supercharging the engine. Using the gas as a mixture in a diesel engine results in derating the engine by only 10% because of its high excess in the gas-air ratio. However, the diesel engine will require a small amount of diesel fuel for ignition. Although gasifier units can be relatively simple for small-scale operations designed, large-scale systems are most efficient. Thus, about 11.4 kcal of woodfuel is required to produce 1 kcal of gas. If the gas is cleaned, then the net return is diminished. The input : output results in an energy return in terms of wood to gas of 1 : 0.09. The equipment for cleaning the gas is expensive and uneconomical for use in rural areas, especially in developing countries. In addition to using the produced gas for internal combustion engines, it may be utilized as feedstock for various chemical products.

C. Pyrolysis Air-dried wood or other biomass heated in the absence of oxygen can be converted into oil, gas, and other valuable fuels. The biomass feedstock, before it is fed to the pyrolysis reactor, must be ground or shredded into smaller than 14-mesh size units. Flash pyrolysis takes place at 500◦ C and under high pressure (101 kPa). After processing the solid char is separated from the fluids produced in a cyclone separator. The char is then used as a heating source for the reactor. Using dry municipal refuse, the resulting products from a kilogram of biomass are water, 10%; char, 20% (energy content is about 4500 kcal/kg); gas, 30% (energy content is 3570 kcal/m3 ); and oil, 40% (energy content is 5950 kcal/kg). Other investigators have reported up to 50% oil production. This gas and oil can be reprocessed, cleaned, and utilized in internal combustion engines. The oil and gas yield from a rapid processing pyrolysis plant is about 37% or about 2.7 kcal return per kilocalorie invested. Since the plant analyzed in the study was processing city wastes, there was no energy or economic charge for biomass material. However, if tropical drywood is used for pyrolysis about 5 kcal of wood is required to produce 1 kcal of oil. The gas from a gasifier-pyrolysis reactor can be further processed to produce methanol. Methanol is useful as a liquid fuel in suitably adjusted internal combustion engines. Employing pyrolysis in a suitably large plant to produce methanol would require at least 1250 t of dry biomass per day. Based on tropical dry-wood, about 32 kcal of wood is needed to produce 1 kcal of methanol (or 1 t of wood yields 14 l of methanol). A more recent study reports that 1 t of wood yields 370 l of methanol. In either case, more than 150,000 ha of forest would be needed to supply one plant. Biomass generally is not available in such enormous quantities from extensive forests and at acceptable prices. If methanol from biomass was used as a substitute for oil (33 quads) in the United States, about 1000 million hectare of forest land per year would be needed to supply the raw material. This land area is much greater than the 162 million ha of United States cropland now in production. Although methanol production from biomass may be impractical because of the enormous size of the conversion plants, it is significantly more efficient than ethanol production using corn based on energy output and economic use of cropland.

D. Vegetable Oil Processed vegetable oils from sunflower, soybean, rape, and other plants can be used in diesel engines. One major

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168 advantage of burning vegetable oils in a diesel engine is that the exhaust smells like cooking popcorn. However, the energetics and economics of producing vegetable oils for use in diesel engines are negative. Sunflower seeds with hulls have about 25.5% oil. The average yield of sunflower seeds is 1560 kg/ha, and in terms of oil this amounts to 216 l of vegetable oil produced per hectare. This much oil has an energy value of 1.7 million kilocalories which appears promising. However, the energy input to produce this yield of 1560 kg/ha is 2.8 million kcal. Therefore, 65% more fossil energy is used to produce a liter of vegetable oil than the energy potential of the sunflower oil. A liter of vegetable oil sells for at least $2 whereas a liter of gasoline at the pump today sells for 40c/ per liter. There is no way that vegetable oil will be an economic alternative to liquid fuels in the future. E. Electricity Although most biomass will continue to be used for cooking and heating, it can be converted into electricity. With a small amount of nutrient fertilizer inputs, an average of 3 t (dry) of woody biomass can be sustainably harvested per hectare per year, although this amount of woody biomass has a gross energy yield of 13.5 million kilocalories (thermal). The net yield, however, is lower because approximately 33 l of diesel fuel per hectare is expended for cutting and collecting wood for transport. This assumes an 80-km roundtrip between the forest and the electric plant. The economic benefits of biomass are maximized when the biomass is close to the processing plant. In addition, a small amount of nitrogen fertilizer has to be applied. For bolewood, 1 t contains about 15 kg of N. Thus about 837,000 kcal is required for 3 t of bolewood. The energy input : output ratio for the system is calculated to be 1 : 6. The cost of producing a kilowatt of electricity from woody biomass ranges from 7–10c/ . This is competitive with other electricity production systems that presently have an average cost of 6.9c/ with a range of 5–13c/ per kWh. Approximately 3 kcal of thermal energy is expended to produce 1 kcal of electricity. Woody biomass could supply the nation with about 5 quads of its total gross energy supply by the year 2050 with the use of approximately 112 million hectare (an area larger than the state of Texas). A city of 100,000 people using the biomass from a sustainable forest (3 t/ha) for fuel would require approximately 220,000 ha of forest area, based on an average electrical demand of 1 billion kilowatthours (860 kcal = 1 kWh). More than 70% of the heat energy produced from burning biomass is lost in its conversion into electricity; this is similar to losses experienced in coal-fired plants. The forest area required to

Biomass Utilization, Limits of

supply this amount of electricity is about the same as that required to supply food, housing, industry, and roadways for a population of 100,000 people. There are several factors that limit reliance on woody biomass. Some have proposed culturing fast-growing trees in a plantation system located on prime land. These yields of woody biomass would be higher than the average of 3 t/ha and with large amounts of fertilizers and freshwater yields might be as high as 15 t/ha. However, this is unrealistic because this land is needed for food production. Furthermore, such intensely managed systems require additional fossil fuel inputs for heavy machinery, fertilizers, and pesticides, thereby diminishing the net energy available. In addition energy is not the highest priority use of forest wood, but rather for lumber for building and pulp. The conversion of natural forests into plantations will increase soil erosion and water runoff. Continuous soil erosion and degradation will ultimately reduce the overall productivity of the land. If natural forests are managed for maximal biomass energy production, loss of biodiversity can be expected. However, despite serious limitations of plantations, biomass production could be increased using agroforestry technologies designed to protect soil and conserve biodiversity.

IV. BIOMASS AND THE ENVIRONMENT The presence of biomass on the land protects not only the land it covers, but also the natural interactions among all species that inhabit the ecosystem. Conversely, the removal of biomass for all purposes, but most especially for energy production, threatens the integrity of the entire natural ecosystem. A. Soil Erosion Once the biomass vegetation has been removed from the land area and the land is exposed to wind and rainfall energy, erosion is a major threat. Land degradation by soil erosion is of particular concern to agriculturists and foresters because the productivity of the soil is diminished. Too often soil erosion and the resulting degradation goes unnoticed (note, 1 mm of soil weighs 15 t/ha). Soil reformation is exceedingly slow. Under agricultural conditions, approximately 500 years (range from 200 to 1000 years) are required to renew 2.5 cm (340 t) of topsoil. This soil formation rate is the equivalent of about 1 t/ha/yr. Forest soil re-formation is slower than in agriculture and is estimated to take more than 1000 years to produce 2.5 cm of soil. The adverse effect of soil erosion is the gradual loss of productivity and eventually the abandonment of the land for crop production.

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Serious soil erosion occurs on most of the world’s agriculture, including the United States where erosion on cropland averages 13 t/ha/yr. In developing countries, soil erosion is approximately 30 t/ha/yr. The rates of erosion are intensifying in developing countries because of inefficient farming practices and because large quantities of biomass are removed from the land for cooking and heating. Rural people who are short of affordable fuels are now being forced to remove crop residues and utilize dung for cooking, leaving their soils unprotected and susceptible to wind and water erosion. Indeed soil erosion caused by wind and water is responsible for the loss of about 30% of the world cropland during the past 40 years. For example, the rate of soil loss in Africa has increased 20-fold during the past 30 years. Wind erosion is now so serious in China that Chinese soil can be detected in the Hawaiian atmosphere during the Chinese spring planting period. Similarly, soil eroded by wind is carried from Africa to Florida and Brazil. Erosion diminishes crop productivity by reducing the water-holding capacity of the soil and reduces water availability to the plants. In addition, soil nutrient levels and organic matter are carried away with the eroding soil and soil depth is lessened. Estimates are that the continuing degradation of agricultural land will depress world food production from 15–30% by the year 2020. Others project that Africa will be able to feed only 40% of its population in 2025 both because of population growth and soil infertility in vital cropland areas. B. Forest Land Erosion Forestlands lose significant quantities of soil, water, and soil nutrients wherever trees are cut and harvested. For instance, the surface water runoff from a forested watershed after a storm averaged 2.7% of the precipitation, but after forest cutting and/or farming water runoff rose to 4.5 percent. In addition, soil nitrogen leached after forest removal was 6 to 9 times greater than in forests with normal cover. Also, the procedures used in harvesting timber and pulpwood biomass contribute to increased erosion because they expose the soil to wind and rainfall energy. Typically, tractor roads and skid trails severely disturb 20–40% of the soil surface in forests. In addition, the heavy equipment needed to harvest and clear the land compacts the soil, resulting in greater water runoff. For example, compaction by tractor skidders harvesting Ponderosa pine reduced growth in pine seedlings from 6 to 12% over a 16-year period. Following clearing, water percolation in the wheel-rutted soils was reduced for 12 years and in log-skid trails for 8 years. This resulted

in a lack of water for the remaining vegetation and limits continual forest biomass production. C. Nutrient Losses and Water Pollution Rapid water runoff and nutrient losses occur when crop biomass residues are harvested for fuel and rainfall easily erodes soils. Water quickly runs off unprotected soil because raindrops free small soil particles that, in turn, clog holes in the soil and reduce water infiltration. This water runoff transports soil organic matter, nutrients, sediments, and pesticides to rivers and lakes where it harms natural aquatic species. For example, conventional corn production lost an average of about 20 t/ha/yr of soil compared with only about 5 t/ha/yr with ridge- and no-till. As mentioned, the water-holding capacity and nutrient levels of soils are lessened when erosion occurs. With conventional corn production, erosion reduced the volume of moisture in the soil by about 50% compared with no-till corn culture. In contrast, soil moisture volume increased when corn was grown in combination with living mulches. Estimates are that about $20 billion in fertilizer nutrients are lost annually from United States agriculture because of soil erosion. Large quantities of nutrients are also lost when fuelwood and crop residues are also removed and then burned. On average, crop residues contain about 1% nitrogen, 0.2% phosphorus, and 1.2% potassium. When burned, the nitrogen is released into the atmosphere. Although some phosphorus and potassium are retained in the ashes, an estimated 70–80% of these nutrients is lost when the fine particulate matter is dispersed into the air during burning process. Thus, only a small percentage of the nutrients in crop residues are conserved even when returning the ash residues to the cropland. D. Water Use All biomass vegetation requires and transpires massive amounts of water during the growing season. Agriculture uses more water than any other human activity on the planet. Currently, 65% of the water removed from all sources worldwide is used solely for irrigation. Of this amount, about two-thirds is consumed by plant life (nonrecoverable). For example, a corn crop that produces about 8000 kg/ha of grain uses more than 5 million liters per hectare of water during its growing season. To supply this much water to the crop, approximately 1000 mm of rainfall per hectare, or 10 million l of irrigation, is required during the growing season. The minimum amount of water required per capita for food production is about 400,000 l/yr. If the water

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170 requirements for biomass energy production were added to this, the amount of required water would be more than double to about 1 million l/yr. In addition to the unpredictable rainfall, the greatest threat to maintaining adequate fresh water supplies is depletion of the surface and groundwater resources that are used to supply the needs of the rapidly growing human population. Aquifers are being mined faster than the natural recharge rate and surface water is also not always managed effectively, resulting in water shortages and pollution that threaten humans and the aquatic biota that depend on them. The Colorado River, for example, is used so heavily by Colorado, California, Arizona, other states, and Mexico, it is usually no more than a trickle running into the Sea of Cortes. E. Air Pollution The smoke produced when fuelwood and crop residues are burned is a pollution hazard because of the nitrogen, particulates, and other polluants in the smoke. A report indicated that although only 2% of the United States heating energy comes from wood, and about 15% of the air pollution in the United States is caused by burning wood. Emissions from wood and crop-residue burning are a threat to public health because of the highly respirable nature of the 200 chemicals that the emissions contain. Of special concern are the relatively high concentrations of potentially carcinogenic polycyclic organic compounds and particulates. Sulfur and nitrogen oxides, carbon monoxide, and aldehydes are also released, but with wood there are usually smaller quantities than with coal.

V. SOCIAL AND ECONOMIC IMPACTS In the future, if the world biomass is used as a major source of the world energy supply, shifts in employment and increases in occupational health and safety problems can be expected. Total employment would be projected to increase 5% if about 11% of the United States energy needs were provided by biomass. This labor force would be needed in agricultural and forest production to plant, cut, harvest, and transport biomass resources and in the operation of various energy conversion facilities. The direct labor inputs for wood biomass resources are 2–30 times greater per million kilocalorie than coal. In addition, a wood-fired steam plant requires 2–5 times more construction workers and 3–7 times more plant maintenance and operation workers than a coal-fired plant. Including the labor required to produce corn, about 18 times more labor is required to produce a million kilocalories of ethanol than an equivalent amount of gasoline.

Biomass Utilization, Limits of

Associated with the possibilities of increased employment are greater occupational hazards. Significantly more occupational injuries and illnesses are associated with biomass production in agriculture and forestry than with either coal (underground mining), oil, or natural gas recovery operations. Agriculture and forestry report 61% more occupational injury and illness rates than mining. In terms of a million kilocalories of output, forest biomass has 14 times more occupational injuries and illnesses than underground coal mining and 28 times more than oil and gas extraction. Clearly, unless safe harvesting practices and equipment are developed and used, increased forest harvesting and agricultural production for energy will result in high levels of occupational injuries and increased medical expenditures and workman compensation. The future development of major biomass energy programs will require large amounts of cropland suitable for biomass production and ultimately result in increased prices for some consumer commodities. The use of commodities, especially grains, for energy leads to competition with traditional uses of these commodities. Thus, with increased grain use for ethanol production, inflation of farm commodity prices could result. This in turn would increase farmland prices and make it more difficult for new farmers to enter the business and for existing small farmers to cope with higher rents, taxes, interest payments, and production costs. Food prices in supermarkets would be expected to increase.

VI. CONCLUSION Certainly increased use of biomass as a fuel could provide the United States and the world with more renewable energy. A major limitation of biomass energy production includes the relatively small percentage (average 0.1%) of light energy that is captured by the earth’s plant material. This governs how much biomass can be produced per unit land area. In addition to solar energy, suitably warm temperature conditions, adequate amounts of water, and the absence of pests are essential for plant growth. In North America, for example, plant growth only occurs for approximately three months of the year. In arid regions of the world plant growth is restricted only to periods of adequate rainfall. The removal of biomass, such as crop residues, from the land for energy production intensifies soil erosion, water runoff, and soil nutrient losses. In addition, the conversion of natural ecosystems into energy-crop plantations would alter and/or reduce the habitat and food sources for wildlife and biodiversity. At present, about half of the world’s biomass is harvested as food and forest products. Thus, there is a limit

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as to how much biomass can be harvested as an energy source without further causing the extinction of more plants, animals, and microbes because of biomass resources on which biodiversity depends. Agriculture and managed forests occupy approximately 70% of the total land area and use about 70% of the total water consumed by society, and this further limits natural biodiversity. However, opportunities do exist to combine agriculture and forest production. If this is to be done several changes would have to be made in many technologies now used in agriculture and forestry. These technologies include conserving soil, water, and nutrient resources. Of particular importance is keeping the land covered with vegetation and maintaining high levels of organic matter in the soil. Although biomass resources have a lower sulfur content than oil and coal, biomass energy conversion and use has associated environmental and public health problems. For example, the chemical emissions from wood-burning for cooking and heating produce serious chemical pollutants, including some carcinogens and other toxicants. In addition, on the basis of a million kilocalorie output, harvesting forest biomass energy is about 14 times more hazardous than coal and oil mining. Ethanol production using grains and other food material for gasohol can be expected to have a significant negative impact on social and economic systems. A major ethanol program would help fuel inflation by raising food prices to the consumer. In addition, “burning food” as ethanol in automobiles has serious political and ethical considerations. In conclusion, the conversion of biomass to provide an energy source has some potential to contribute to world energy needs, but the associated environmental, health, social, and economic problems must be carefully assessed. The foremost priority is the supply of food. Especially vital to this goal is maintaining an ample supply of fertile cropland needed to feed the rapidly growing world population.

ACKNOWLEDGMENT I sincerely thank the following people for reading an earlier draft of this article and for their many helpful suggestions: Andrew R. B. Ferguson, Optimum Population Trust, U.K.; Marcia Pimentel, Division of Natural

Sciences, Cornell University; Joel Snow, Iowa State University; and Paul Weisz, Pennsylvania State University.

SEE ALSO THE FOLLOWING ARTICLES BIOREACTORS • ENERGY FLOWS IN ECOLOGY AND IN THE ECONOMY • GREENHOUSE EFFECT AND CLIMATE DATA • POLLUTION, AIR • POLLUTION CONTROL • RENEWABLE ENERGY FROM BIOMASS • WASTE-TO-ENERGY SYSTEMS • WATER POLLUTION

BIBLIOGRAPHY Ellington, R. T., Meo, M., and El-Sayed, D. A. (1993). “The net greenhouse warming forcing of methanol produced from biomass,” Biomass Bioenergy 4(6): 405–418. Ferguson, A. R. B. (2000). “Biomass and Energy,” The Optimum Population Trust, Manchester, U.K. Pimentel, D. (1991). “Ethanol fuels: Energy security, economics, and the environment,” J. Agr. Environ. Ethics 4, 1–13. Pimentel, D., Doughty, R., Carothers, C., Lamberson, S., Bora, N., and Lee, K. “Energy inputs in crop production in developing and developed countries,” J. Agr. Environ. Ethics, in press. Pimentel, D., and Kounang, N. (1998). “Ecology of soil erosion in ecosystems,” Ecosystems 1, 416–426. Pimentel, D., and Krummel, J. (1987). “Biomass energy and soil erosion: Assessment of resource costs,” Biomass 14, 15–38. Pimentel, D., and Pimentel, M. (1996). “Food, Energy and Society,” Colorado University Press, Boulder, Colorado. Pimentel, D., and Strickland, E. L. (1999). “Decreased rates of alluvial sediment storage in the Coon Creek Rasin, Wisconsin, 1975–93,” Science 286, 1477–1478. Pimentel, D., Rodrigues, G., Wang, T., Abrams, R., Goldberg, K., Staecker, H., Ma, E., Brueckner, L., Trovato, L., Chow, C., Govindarajulu, U., and Boerke, S. (1994). “Renewable energy: economic and environmental issues,” BioScience 44, 536–547. Pimentel, D., Warneke, A. F., Teel, W. S., Schwab, K. A., Simox, N. J., Ebert, D. M., Baenisch, K. D., and Aaron, M. R. (1988). “Food versus biomass fuel: Socioeconomic and environmental impacts in the United States, Brazil, India, and Kenya,” Adv. Food Res. 32, 185–238. Shapouri, H., Duffield, J. A., and Graboski, M. S. (1995). “Estimating the Net Energy Balance of Corn Ethanol,” Agricultural Economic Report, Washington, DC. Tripathi, R. S., and Sah., V. K. (2000). A biophysical analysis of material, labour and energy flows in different hill farming systems of Garhwal Himalaya, “Agriculture, Ecosystems and Environment,” in press. WHO (1996). “Micronutrient Malnutrition—Half of the World’s Population Affected,” No. 78, 1–4, World Health Organization.

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Biomass, Bioengineering of Bruce E. Dale Michigan State University

I. II. III. IV. V.

Background Characteristics of Biomass Uses of Biomass Bioprocessing of Biomass Potential and Limitations of Biomass and Biobased Industrial Products

GLOSSARY Biomass Plant material. Bioprocessing Any chemical, thermal, physical or biological processing done to biomass to increase its value. Biobased industrial products Plant-derived chemicals, fuels, lubricants, adhesives, plastics—any and all industrial products derived from biomass that are not used for human food or animal feed. For purposes of this article, biomass is bioprocessed into biobased industrial products. Biorefineries Large, highly integrated facilities, analogous to petroleum refineries, that process biomass to biobased industrial products and other value-added products. Life cycle analyses Comprehensive inventories of the material and energy flows required to produce, use and dispose of specific products throughout their entire life cycles. Lignocellulose The structural portion of most plants, composed of a complex mixture of cellulose, hemicellulose and lignin and comprising the vast majority of all biomass. Cellulose is a polymer of glucose

(sugar) while hemicellulose is a polymer made up of a variety of sugars. Lignin is a complex polymer of phenylpropane units. Sustainable development Economic development that meets the legitimate needs of current generations without compromising the ability of future generations to meet their own needs. BIOMASS is the only potentially renewable source of organic chemicals, organic materials and liquid transportation fuels. The biomass resource is huge. While estimates are necessarily imprecise, it is believed that photosynthesis fixes approximately 150 billion tons of new plant matter annually on the planet. Production of biobased industrial products has the potential to benefit both the economy and the environment and to provide new pathways for sustainable economic development. The energy value of our renewable plant resource is approximately ten times the total energy value of all other forms of energy used by humanity including all fossil fuels, hydropower, nuclear energy and so on. Biomass is also relatively inexpensive and compares favorably with petroleum on a cost per pound basis and, frequently, on

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142 a cost per unit of energy basis. Although the biomass resource is huge and comparatively inexpensive, we have invested much less effort in learning how to bioprocess or convert it efficiently to biobased industrial products than we have invested in converting petroleum to meet our needs for fuels, chemicals, materials, and other industrial products. Compared to the petroleum processing industry, the biomass processing industry is still relatively underdeveloped, although the biomass processing industry is in fact already very large and is also growing rapidly. Thus much of this article deals with what is required for the biomass processing industry to grow further and what some of the possible and desirable growth paths for this industry might be.

I. BACKGROUND The potential benefits (including economic, environmental and national security benefits) of obtaining a larger fraction of our fuel and chemical needs from biomass rather than from petroleum have driven increasing interest in biobased industrial products in the United States and many other countries. Lack of cost–effective bioprocessing technology is perhaps the principal barrier to more economical production of biobased industrial products. Although biomass is abundant and low cost, unless we learn how to cost-effectively convert biomass to these industrial products, their potential benefits will be largely unrealized. While the potential benefits of biobased products are certainly real, it is also correct that unless such products are produced with proper intelligence and care, their benefits may be reduced or even negated. We must be careful that biomass is grown, harvested, converted to industrial products, and that these products are used and disposed of, in sustainable, environmentally sound systems. Careful, thorough and easily verified life cycle analyses will help us realize the potential of biobased industrial products to benefit our economy and our environment and also to avoid potential problems with the production and use of these products. One of the most important areas demanding careful life cycle (whole system) attention for biomass conversion to industrial products is the potential conflict with food and feed production. Biomass production for biobased industrial products seems to conflict with use of the same agricultural resources for human food and animal feed. This article briefly addresses this crucial point and finds considerable room for optimism.

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II. CHARACTERISTICS OF BIOMASS A. Production of Biomass 1. Natural Inputs to Biomass Production Natural (or ecosystem) inputs to biomass production are soil (including the associated nutrients and living organisms found in soil), genetic information, air, water, and sunlight. All of these inputs are potentially renewable indefinitely with proper oversight and intelligent design. In fact, biomass production has the potential to improve soil, water, and air quality. The entire life cycle of biomass production, bioprocessing, and biobased product use and disposal should be examined carefully to discover and properly exploit such opportunities. Intelligent design of biomass processing systems should take advantage of opportunities to improve the environment and enhance ecosystem stability under circumstances peculiar to each region and product. With careful and thoughtful design, biomass production and processing can increase or enhance the “natural capital” of soil, air, and clean water upon which all life depends. Human inputs to biomass production include additional plant nutrients beyond those provided through the ecosystem, plant genetic improvement, labor, financial capital and intelligence, as referred to above. Much agriculture is also practiced with large inputs of fossil fuels. As mentioned, thorough and careful life cycle analysis is required to determine whether biomass processing to biobased products actually fulfils its potential to give us a more sustainable economy. 2. Potential and Actual Yields of Biomass A key factor determining the economic (and therefore the resulting ecological) benefits of biomass production and processing is the yield of biomass, defined as the annual production of biomass (dry weight) per unit land area, often expressed as tons of dry biomass per acre per year. Meeting legitimate human needs by more intensively producing biomass (i.e., increasing yields) will allow larger tracts of land to be set aside for recreation, parks, and biological reserves. Biomass yields vary widely. The upper limit of solar energy conversion efficiency by biomass appears to be about 12% (incoming solar energy converted to the energy content of plant material). Yield seems to be tied closely to conversion efficiency; the higher the conversion efficiency, the higher the yield. Sugarcane is one of the more efficient crops, with solar energy capture efficiencies in the neighborhood of 2 to 3% and corresponding biomass yields of between 25 and 35 dry tons per acre per year. The corresponding efficiency value for corn is about 0.8%.

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FIGURE 1 U.S. land required for biomass energy.

Increasing biomass yields is a crucial area for research. We have invested much effort and money in increasing the yields of grains such as corn. Average per acre corn yields increased at a rate of over 3% per year between 1950 and the present: corn yields were about 28 bushels per acre per year in 1947 and topped 127 bushels per acre per year in 1997. However, we have done comparatively little genetic or agronomic research to increase the yields of the perennial grasses and tree crops on which a sustainable biomass processing industry will likely be based. Thus there is great room for improving these yields. Biomass is currently the most practical collector we have of solar energy on a large scale. The solar energy incident on the United States each year is about 600 times our annual energy consumption of about 95 quads (one quad equals one quadrillion BTU or one million billion BTU). The higher the biomass yields, the more solar energy is collected per unit land area. At a solar energy conversion efficiency of 0.8% (corn efficiency), approximately 40% of the U.S. land area placed in continuous crop production would produce biomass with an energy value equal

to our total use of energy from all forms. At this efficiency, about 10% of our land area, or 100 million acres, would be required to produce the energy equivalent of all of the petroleum we use. This is roughly equal to the land currently in hay production (60 million acres) plus land idled under government programs. Obviously, other inputs in addition to solar energy are required for biomass production. Nonetheless, these statistics gives some idea of the potential to meet our energy needs from biomass. Figure 1 summarizes some of figures for U.S. land area usage and the area required to equal our energy usage at solar energy conversion efficiencies typical of corn and sugar cane. 3. Comparison of Biomass and Petroleum Worldwide consumption of crude oil, a large but nonetheless limited resource, was about 27 billion barrels per year in 1999 or about 4 billion tons, with an approximately equal amount of coal consumed. As mentioned earlier, total production of new biomass, an indefinitely renewable resource, is approximately 150 billion tons per year.

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144 The energy value (heat of combustion) of petroleum is about twice that of biomass (trees have a higher energy content than grasses) while coal averages about one and a half times the energy value of biomass. The lower energy value of biomass is due to the fact that it contains substantial oxygen, while petroleum has little or no oxygen. The lower energy content (i.e., the higher oxygen content) of biomass is both an advantage and a disadvantage for this renewable resource. Biomass and the many oxygenated compounds that can be made from biomass are inherently more biodegradable and therefore more environmentally compatible than petroleum and petroleumderived compounds. Put another way, a large spill of wheat straw is not an environmental disaster, while we are well aware of the impacts of petroleum spills. Powerful economic considerations tied to raw material use efficiency also direct us toward maintaining the oxygen molecules in biobased industrial products. Petroleum, a liquid, is easier and less expensive to transport and store than solid biomass. One consequence of this fact is that much biomass processing will likely be done relatively close to where the biomass is produced. This may provide opportunities to integrate biomass production with biomass processing and to more easily return to the land the unused or unusable components of biomass. Few such opportunities exist with petroleum processing. In many climates, biomass production takes place only during part of the year, so there are additional storage issues that are unique to biomass. Finally, large quantities of biomass can be produced in many, if not most, countries, while comparatively few countries produce significant quantities of petroleum. Thus biomass production is inherently more “democratic” than petroleum production and is certainly less susceptible to political manipulation. 4. Cost of Biomass versus Fossil Feedstocks Petroleum costs varied between about $10 and $20 per barrel ($65 to $130 per ton) during the decade of the 1990s. Currently oil prices are about $30 per barrel or roughly $200 per ton. Coal is available for approximately $30 per ton. By comparison, corn at $2.50 per bushel, an “average” corn price over the last decade, is roughly equivalent to $90 per ton. Corn is currently less than $2.00 per bushel, or about $70 per ton, approximately one third the current price of crude oil. Hay crops of different types and qualities are available in very large quantities (tens of millions of tons) for approximately $30–$50 per ton and several million tons per year at least of crop residues such as rice straw and corn stover are available in the United States for less than $20 per ton. Figure 2 summarizes some of these comparisons of the relative prices of biomass and fossil

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resources. Worldwide, many hundreds of millions of tons of crop residues such as rice straw, sugar cane bagasse and corn stover are likely to be available at very low cost, probably less than $20 per ton. Thus while fossil resources are relatively inexpensive (even given oil price volatility) renewable plant resources are equally inexpensive, and in many cases, less expensive. The importance of this fact to biomass processing cannot be overstated. Plant raw material costs are crucial for the development of cost-competitive biobased products. For well-developed processes making commodity chemicals and fuels, approximately 50–70% of the total production costs are due to the raw material costs. Thus inexpensive biomass should eventually lead to inexpensive biobased products, if the necessary bioprocessing technologies for converting biomass to biobased products can also be made inexpensive. In general, we do not yet have inexpensive biomass processing technology. However, if the necessary research and development work is done to learn how to inexpensively convert biomass to biobased products, there is every reason to believe that these biobased products can compete on a cost and performance basis with similar products derived from petroleum. To illustrate, the large chemical companies Dow Chemical and DuPont have recently announced plans to produce monomers for polymer (plastic) production from renewable sugars and starches. These carbohydrates are relatively inexpensive, and the companies have also developed inexpensive and effective conversion technologies to produce the monomers. For example, Cargill Dow Polymers (CDP) LLP (Minnetonka, MN) is building the first of up to five large processing plants to convert corn starch into lactic acid and then into polymers (polylactides). Although biodegradability of the polymers is obviously a benefit, CDP expects its polylactide polymers to compete on a cost and performance basis with petroleum-derived competing products. Similarly, DuPont’s carbohydratederived product, 1,3 propanediol, is intended to compete directly with the identical molecule produced from petroleum. The chemical industry is therefore beginning to change its raw material base. As technologies improve and bioprocessing costs decrease, there is every reason to believe that more such products will follow.

B. Major Types of Biomass: Their Production and Composition 1. Sugar Crops The major sugar crops are sugar cane and sugar beets. Worldwide, approximately 100 million tons per year of sugar (sucrose) are produced from sugar cane and sugar beets. Most of these sugars are used ultimately in human

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FIGURE 2 Cost of fossil vs. biomass feedstocks.

nutrition. Sugar cane is grown largely in the tropics while sugar beets are grown mostly in temperate zones. Approximately 10 dry tons of a fibrous residue (called bagasse) are produced for every ton of cane sugar while about 0.5 dry tons of residue are produced for every ton of beet sugar. Worldwide the total production of sugarcane bagasse is approximately 800 million metric tons per year. These residues have few other uses and represent a very large potential resource for bioprocessing to biobased industrial products. Sugar cane bagasse consists of approximately 40% cellulose, 30% hemicellulose and 20% lignin with small amounts of minerals, sugars, proteins and other compounds. The composition of bagasse is, however, variable depending on growing conditions, harvesting practices and processing methods. Beet sugar residue consists of approximately equal portions of cellulose, hemicellulose and pectins, with a few percent of lignin. Cellulose and hemicellulose are polymers of glucose and other sugars. However, the sugars in cellulose, and to a lesser degree those in hemicellulose, are not very good animal feeds and they are essentially indigestible as human foods.

Microbial and enzymatic conversion of these sugars in hemicellulose and cellulose to biobased products is also significantly limited for the same reasons cellulose and hemicellulose are not easily digested by animals. The resistance of cellulose to conversion to simple sugars is a key limitation in biomass processing that must be overcome if biomass processing is to attain its full potential. To put the potential of cellulose and hemicellulose in perspective, the potential sugar that might be produced from cellulose and hemicellulose in sugar cane bagasse alone is approximately six times the total sugar produced worldwide by both sugar cane and sugar beets. 2. Starch Crops A wide variety of starch crops (mostly grains) are grown worldwide, including corn (maize), rice, wheat, manioc (cassava), barley, rye, potatoes and many more. At least 2 billion tons per year of these crops are produced worldwide. While most sugar is used to feed human beings, much grain is used to feed animals, particularly in the more developed countries. One key indicator of a country’s

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146 economic growth, in fact, is a substitution of meat for grains in the human diet. Animals convert grain to meat or milk with widely varying efficiencies, however. Fish, poultry, and swine are relatively efficient converters, while beef cattle are considerably less efficient. Most grain crops produce a byproduct, or residue, that is primarily composed of cellulose, hemicellulose, and lignin, called collectively lignocellulose. Thus very large tonnages of rice straw, corn straw (called corn stover), and many other straws are produced as a low value (and low cost) byproduct of grain production. Approximately 1 to 2 tons (dry weight) of these straws are produced per dry ton of grain. Using this ”rule of thumb,” the total worldwide production of just corn stover and rice straw is in the neighborhood of 1 billion tons per year. Taken together with sugar cane bagasse production, the total amount of corn stover, rice straw and bagasse produced each year is approximately 2 billion tons. Very large quantities of other straws and crop processing residues are also produced. Many such residues are produced at centralized processing facilities. While some of this residual plant matter should be left on the field, much of it can be removed and used elsewhere without degrading soil fertility. For example, rice straw is often burned to clear the fields for the next crop. There is considerable political pressure in the United States and elsewhere to eliminate or greatly reduce this practice of field burning. 3. Plant Oil and Protein Crops There are many different plant oil crops including soybeans, palm, coconut, canola, sunflower, peanut, olive and others. The total worldwide production of fats and oils by these crops exceeds 75 million tons per year, with an additional 12 million tons per year or so of animal fats. (An oil is simply a liquid fat.) Most plant oils go into human foods or animal feeds. However, there is a very long history of also using and modifying plant oils for fuels, lubricants, soaps, paints and other industrial uses. Oils consist chemically of an alcohol (glycerol) to which are attached three long chain carboxylic acids of varying composition. Plant oil composition varies widely with species and the composition strongly affects the industrial uses to which these oils can be put. Therefore by modifying these oils, they can potentially be tailored to desired applications. The other major product of oilseed crops is a high protein “meal,” usually produced by expelling or extracting the oil from the seed. Total world production of high protein meals from oilseeds is approximately 180 million tons per year. The predominant oilseed meal is soybean meal containing approximately 44% protein. While there are some industrial uses for this meal, the bulk of it is fed to animals.

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As with the starch crops, most of these oilseed crops produce one or more residues that are rich in lignocellulose. For example, soybean straw is typically left in the fields when the beans are harvested. Soybean hulls are produced as “wastes” at the oilseed processing plant. In the United States, approximately 10 million tons per year of these soybean hulls are produced as a byproduct of soybean crushing operations. 4. Tree and Fiber Crops In contrast with the crops mentioned, essentially all of the wood harvested is destined for industrial uses, rather than food/feed uses. Production of wood for lumber in the United States amounts to about 170 million tons per year while U.S. pulpwood production (destined for all kinds of paper uses) is about 90 million tons/year. A wide variety of industrial chemicals such as turpentine, gums, fats, oils, and fatty acids are produced as byproducts of pulp manufacture. Not all paper is derived from trees, however. Some grasses and crop residues such as kenaf and sugar cane bagasse have been used or are being considered as fiber/paper crops. The giant reed kenaf, in particular, has very rapid growth rates and favorable agronomic characteristics. A major impediment to its introduction as an alternative newsprint crop seems to be the huge capital investment required for a new pulp and paper plant. The growing worldwide demand for paper products of all kinds may limit the ability to use tree and pulpwood crops for other industrial applications, given the value of long plant fibers in paper production. Even short rotation woody crops (trees grown for energy use as if they were grasses), must cope with the demand for that land and the long fibers grown on it for pulp and paper uses. Typical pulp prices are in the neighborhood of $600 per ton or $0.30 per pound, a high raw material cost hurdle indeed for commodity chemicals that are often targeted to sell for less than $0.30 per pound. Some residues from fiber crop production and processing may be available at much lower cost and could perhaps be used for chemical and fuel production. Typically these residues are burned to get rid of them and recover at least their energy value. The most important fiber crop is cotton. Worldwide production of cotton in 1998 totaled about 91 million bales, each weighing about 480 lb. Given the high value textile uses of cotton, it is similarly unlikely that much cotton will be devoted to other uses. However, there are many millions of tons of wastes generated at cotton gins and mills that might be used industrially if appropriate, low-cost, conversion technologies were available. Chemically, these tree and fiber crops and their residues are essentially all

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lignocellulosic materials, i.e., they are composed mostly of sugar polymers and lignin. 5. Forage and Grass Crops For purposes of this article, we will not distinguish between grasses and legumes, but will consider all nonwoody annual and perennial plants as “grasses.” Most grasses utilized by humans are employed for animal feeding. Most forage grasses are also produced for local use and are not widely traded, making it difficult to establish a “market price” for many grasses. Available statistics on forage and grass crops are much less complete than for the sugar, starch and oilseed crops. However, there are approximately 7 billion acres worldwide devoted to animal pasture. If we assume that only 1 ton of forage grasses is produced per acre per year on such lands (the U.S. average for managed hay lands is approximately 3 tons per acre per year), the total amount of animal forage from pasturelands is about 7 billion tons per year, on a worldwide basis. In the United States we produce at least 300 million tons per year of mixed forage grasses (dominated by alfalfa). Forages and grasses vary widely in composition, although they can be considered lignocellulosic materials.

However, grasses contain a much wider variety of components that do most tree species. In addition to cellulose, hemicellulose and lignin, grasses often contain 10% or more of protein, in addition to minerals, starch, simple sugars, vitamins and other components. The wider variety of components in grasses versus woody plants offers the potential for producing additional valuable products, but may also complicate the processing required. To summarize this section, in very rough terms the world’s agricultural system produces about 2.5 billion tons per year of total sugar, starch, oil, and plant protein to feed both humans and animals, as well as for some industrial uses. At least this much crop residue is also produced as a byproduct of sugar, starch and oilseed crops. Crop residues are generally lignocellulosic materials. Additionally, well over 10 billion tons per year of lignocellulosic materials are grown with some degree of human involvement as crop and forest residues, animal forages and pasture, not including the managed production of timber and pulpwood. Many more billions of tons of lignocellulosic materials are produced annually in the biosphere with essentially no human intervention. Thus the size of the lignocellulosic resource dwarfs that of the food/feed resource represented by the sugar, grain and oilseed crops. Figure 3 attempts to summarize these data on the annual production of biomass

FIGURE 3 World food and forage production (millions of tons).

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148 for these many food and feed uses, as well as available crop and forestry residues. C. Biotechnology and Biomass Production 1. Modify Biomass Composition for Easier Processing Plant breeding and/or molecular biology techniques can be used to alter the composition of plant matter to make it easier to process. As mentioned previously, given the already relatively low cost of biomass, it is believed that reducing the costs of converting biomass to industrial products will be the key factor in helping these products compete with petroleum-derived products on a much larger scale. For example, reducing the lignin content of grasses and trees should make it easier to convert the cellulose and hemicellulose portions to sugars that might then be fermented or otherwise processed to a wide variety of chemicals and fuels. Changing the fatty acid composition of a particular plant oil could improve the ability to separate that oil in a processing facility. The possibilities are quite literally endless. The ability to modify the raw material composition and properties has no parallel in petroleum refining (or hydrocarbon processing generally). This is a major potential advantage of biobased industrial products that should be exploited whenever possible. 2. Enhance Biomass Yields and Reduce Inputs As mentioned, both plant breeding and molecular biology can be used to increase the yields of biomass grown for industrial uses and to reduce the inputs required to produce these industrial crops. High yields are important both to reduce the costs of biobased products and to decrease the total amount of land required to supply these products. Reductions in crop production inputs such as fertilizers, pesticides, herbicides and even water will also tend to reduce the costs of biomass production and could have very large, positive environmental effects. For example, deep-rooted perennial grass species or trees destined for conversion to industrial products might be planted around fields devoted to row crops such as corn and at the edges of streams to intercept fertilizers and pesticides in groundwater and to reduce soil erosion. Agricultural chemicals in runoff are believed to contribute significantly to oxygendepleted, and therefore life-depleted, regions in the Gulf of Mexico and elsewhere. 3. New Products Breeding has long been used to alter the composition of plant materials, for example to increase the content of

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various starch fractions in corn or to modify the sugar content of sugar cane. Plant breeding has also been used to alter the composition and amounts of various biomass fractions for industrial uses, for example, to increase the resin (rubber) content in the desert shrub called guayule. Such plant breeding efforts are relatively uncontroversial. However, molecular biology/genetic engineering can also be used to modify the existing genes of plants and to transfer entirely new genes into plants. For example, bacterial genes for a biodegradable plastic called have been successfully expressed in plants, leading to the possibility of “chemical plants in green plants.” This is an exciting and technically very promising possibility. It is also a possibility with potentially great environmental benefits. However, considerably more political and environmental controversy is likely to surround such efforts. Careful studies will be needed to demonstrate that expression of foreign genes in plants destined for industrial uses will not lead to undesired environmental or human health consequences.

III. USES OF BIOMASS A. Current Uses 1. Food/Feed Consumption and World Protein/Calorie Demand Average human requirements are approximately 2000 kcal of food energy and about 50 g of protein daily, in addition to smaller amounts of essential oils and vitamins. Assuming a total world population of 6 billion people, then the total human demand for protein is approximately 120 million tons/year and the total calorie requirement is about 4.5 million billion kcal/year (4.5 × 1015 kcal/year). Total world grain (corn, wheat, rice, oats, sorghum, barley, millet and mixed grains) production in 1998/1999 was approximately 2.1 billion tons. If we assume that grain contains on average 70% carbohydrate (sugars) and 11% protein, then this grain crop alone is sufficient to supply all of the calorie needs of the world’s people and about 50% more than the protein needs of all of humankind. If we include the additional calories and protein available from sugar cane and sugar beets, oilseeds and a myriad of other crops such as potatoes and cassava, the total worldwide production of calories and proteins is several fold greater than the human demand. Obviously, much of the plant matter we grow is used to feed animals, not people directly. However, if we so chose, we could easily feed the world’s population with an adequate, plant-based, diet using a fraction of the land now devoted to agriculture and animal husbandry. (There

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FIGURE 4 Human needs for protein and calories vs. nutrient production in crops and lignocellulosics.

is also some consumption of plant matter for industrial uses, the subject of this article.) Past history suggests that people seek to increase their consumption of meat, milk and eggs as their income grows. Looking at these consumption figures in a different light, if we found another way to meet more of the protein and energy (calorie) needs of our animals from sources other than grains and oilseeds, we could then free up large quantities of grain and oilseed crops for other uses, including industrial uses. From the crop production statistics quoted above it is obvious that the potential and actual production of grasses and other lignocellulosic materials far exceeds the production of grains, oilseeds and sugar crops.

2. Animal Feeds In 1998 the United States produced about 40 million tons of beef, pork, and poultry as well as billions of dozens of eggs and tens of millions of tons of milk. To generate these products, livestock and poultry consumed well over 500 million tons of feed expressed on a feeding value equivalent to corn. Over half of this total feed was from

forages, about two thirds of which was grazed as pasture and the rest of which came from harvested forages such as hay and silage. The remainder of animal feed consumed was concentrates such as corn, sorghum, oats, wheat, etc. If it were possible to derive more and better animal feeds from forages and other lignocellulosic materials, it might be possible to increase the use of agricultural raw materials for biobased industrial products without negatively impacting food availability and food prices.

3. Existing Fuels/Chemicals/Materials Uses of Biomass As described above, biomass has long been used as a solid fuel, as a building material and also as a source of fiber for clothing and paper. These uses continue. In the United States, the forest products industry is valued at $200 billion per year and the cotton produced is worth another $5 billion per year. Prior to the early 1800s, biomass was in fact the chief source of fuel and materials. With the coming of the Industrial Revolution, a gradual switch from biomass as the major fuel source took place, first through a transition to coal and later to petroleum and natural gas. The oil

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150 refining industry was developed over about the last 120 years and catalyzed the development of a huge chemical industry, based mostly on petroleum as the ultimate raw material. Today in the United States, the chemical process industries have total sales of over $360 billion per year and the petroleum refining industry is worth about $250 billion per year. Use of biomass for chemicals and materials is relatively small, apart from building materials (wood products). In the United States, more than 90% of total organic chemical production is based on fossil feedstocks. Biomass accounts for less than 1% of all liquid fuels, essentially all of it ethanol derived from corn. Approximately 7% of the total U.S. corn crop is processed into fuel ethanol, industrial starches, industrial ethanol and other chemicals. Not withstanding the relatively small size of the biomassderived chemicals and fuels industry, this industry produces a very wide range of products including oils, inks, pigments, dyes, adhesives, lubricants, surfactants, organic acids and many other compounds. As we have seen, biomass is relatively low cost. However, the processes for converting biomass to industrial products are not, in general, well enough developed or low cost enough to compete effectively with comparable petroleum-derived products. Petroleum-derived products are supported by over a century of research, development and commercial experience. However, the competitive position of biomass is beginning to change. This change is being driven by a combination of technical, economic and social/political factors. Several recent authoritative reports suggest a gradual shift over this next century to a much larger fraction of fuels, chemicals and materials derived from biomass. B. New and Developing Uses of Biomass 1. New Chemicals and Materials Uses From 1983 to 1994, the sales of some biomass-derived products (fuel and industrial ethanol, corn syrups, citric acid, amino acids, enzymes and specialty chemicals, but excluding pharmaceutical products) rose from about $5.4 billion to approximately $11 billion. These products seem likely to continue to grow. The market for new and existing enzymes may be particularly strong, given the ability of enzymes to transform biomass into new products and to provide more environmentally clean chemistries for older petroleum-derived products and processes. Enzymes also have growing applications to improve environmental quality while reducing costs in selected agricultural and domestic uses (e.g., animal feed processing and detergents). Enhanced environmental compatibility

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and economic benefits are also key factors driving the adoption of soybean oil-based inks. These soybased inks were introduced in the 1970s in response to oil shortages and now account for about 40% of all inks. In the United States, approximately 100 million tons per year of organic chemicals are produced annually, with much less than 10% of these chemicals currently derived from biomass. It seems likely that chemical uses of biomass will grow fastest among these or other organic chemicals, particularly for those chemicals that contain oxygen. Some examples of these oxygenated chemicals include organic acids and their derivatives (acetic, adipic, lactic and succinic acids and maleic anhydride), alcohols (butanol, isopropanol, propanediol, and butanediol) and ketones (acetone, methyl ethyl ketone). Indeed, the Cargill–Dow joint venture is focused on polymer production from lactic acid while the DuPont venture with Tate and Lyle is focused on 1, 3 propanediol as another polymer feedstock. Therefore, bioplastics may prove to be the most rapidly growing new materials application for biomass. Industrial starches, fatty acids, and vegetable oils can serve as raw materials for bioplastics, including polymer composite materials. Waste paper and crop and forest wastes and virgin materials are being used as the basis of new composite materials and new fabrics, including Tencel, the first new textile fiber to be introduced in 30 years. It is instructive to consider the amount of plant matter that might be consumed by new chemicals and materials uses of biomass. Given total U.S. production of about 100 million tons of organic chemicals annually, this is about one third of the total mass of the U.S. corn crop of approximately ten billion bushels per year. The corn residue or stover that might be converted to various products to substitute for or replace these organic chemicals is easily equal to the total mass of these organic chemicals, even without converting any of the corn itself. Furthermore, corn yields continue to increase. If we assume a 1% per year increase in yield for corn (versus 3% per year over the past 50 years) and no change in the planted acreage, then the annual increase in corn produced is about 100 million bushels per year, or over 2 million metric tons of new corn every year. The Cargill– Dow Polymers plant being opened in Blair, Nebraska, in late 2001 will produce 140,000 metric tons per year of polylactic acid from approximately 200,000 metric tons of corn. That is, a new large scale plant for bioplastics will only use about 10% of one year’s increase in the corn crop. Thus it seems unlikely that biomass use for chemicals and materials will really have much effect on grain supplies and prices. However, this does not hold true for new large scale liquid fuel uses of biomass.

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2. New Liquid Fuels from Biomass Total consumption of gasoline and diesel fuel in the United States is about 150 billion gallons per year. Assuming an average density of these liquid fuels of six pounds per gallon, the total mass of raw material that would need to be processed just to supply the U.S. with liquid fuel is about 450 million tons per year, assuming complete conversion of the mass of the raw material into fuel. In practice, significantly less than 100% raw material conversion to fuel products will be obtained. In the case of biomass raw materials, however, the situation is even more constrained. If the entire domestic corn crop of about 10 billion bushels per year were converted to fuel ethanol, the total gallons of fuel produced would be less than 20% of domestic diesel and gasoline consumption. However, ethanol has an energy content about 70% of that of gasoline, a consequence of its oxygenated character. Thus a gallon of ethanol will provide lower vehicle mileage than a gallon of gasoline, even when burned in high compression engines designed to take advantage of its high octane value. Thus grain-derived ethanol can never meet more than a small fraction of our liquid fuel needs, however important corn ethanol may be as a bridge to a biomass fuel industry based on lignocellulosics. What is the potential of the lignocellulosics for liquid fuel production? The United States produces at least 300 million tons per year of lignocellulosic crop and forest residues that might be available for conversion to liquid fuels. Current laboratory best yields of ethanol from such residues are about 100 gallons per ton. The total ethanol that might be produced from these lignocellulosic residues is therefore approximately 30 billion gallons per year, a significant fraction of our total domestic liquid fuel demand. Worldwide, if all of the corn stover, rice straw and sugarcane bagasse now produced were converted to ethanol at these yields, approximately 200 billion gallons of ethanol could be generated annually, versus an approximate worldwide consumption of petroleum-derived liquid transportation fuels of 800 billion gallons per year. The requirements for liquid fuels obviously depend strongly on vehicle mileage. For example, if average vehicle fuel efficiency were to increase by a factor of two, demand for liquid fuels would be cut in half. Were that to happen, the fuel ethanol that could be produced just from the residues of these three major crops might satisfy nearly half of the worldwide demand for liquid transportation fuels, without the need to plant additional crops dedicated for energy uses. We must not lose sight of the need to work on the demand side of the fuel equation, as well as the supply side.

3. Land Requirements for Fuel Production However, assuming no improvement in vehicle efficiency and that only half of the tonnage of the three major crop residues (corn stover, rice straw and sugarcane bagasse) is available for conversion to fuel ethanol (because of possible constraints of collection, erosion, alternative uses, etc), the approximate worldwide liquid fuel replacement that might be required from biomass-derived ethanol could be as high as one thousand billion gallons per year. At 100 gallons per ton, roughly 3.0 billion acres of land worldwide would need to be devoted to liquid fuel production from biomass to produce the total required biomass tonnage of 10 billion tons per year, assuming that the average U.S. hay production of 3 tons per acre per year were obtained. However, as pointed out earlier, there is great potential to increase crop yields. Several tropical grasses, including sugarcane, have been shown to yield as much as 25–35 tons of dry matter per acre per year. Furthermore, most land currently used for animal pasture (about 7 billion acres worldwide) is not managed for intensive biomass production. For example, the United States has about 600 million acres in permanent pasture. Grazing animals obtain about 150 million tons of forage annually from this pasture, for an average yield of less than 0.3 tons per acre per year, versus about 3 tons per acre per year from our managed hay lands. Without a demand, there is no incentive to increase forage grass production on the available pasture land. Therefore the demand for land and other agricultural resources required to support biobased industrial products is probably not a factor for chemicals and materials, but will be an issue for biobased fuels. The demand for land to supply liquid fuels depends on the yields of biomass from the land, the yield of fuel from the biomass and the miles traveled per unit of fuel. All three factors are important and must be considered in conjunction. Increasing the efficiency (yield) of each step will increase the overall system efficiency. In particular, biomass conversion to fuel ethanol must be highly efficient and low cost if the product is to compete with petroleum-derived fuels. 4. Cost of Liquid Fuel Production from Biomass It is well known that fuel ethanol derived from corn must be subsidized to compete with gasoline. Raw material costs (primarily the corn) alone are in the neighborhood of $1.00 per gallon of ethanol produced, without any allowance for processing costs. Therefore, it seems unlikely that corn ethanol will ever be able to compete economically with petroleum-derived fuels. Nonetheless, a large

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152 industry producing over 1.3 billion gallons of fuel ethanol from corn in highly integrated, efficient plants has arisen in the United States over the past twenty years, based at least partly on this subsidy. It is not the purpose of this article to argue the pros and cons of subsidizing the corn ethanol industry. However, we note that the existence of the corn ethanol industry provides a learning opportunity, a production platform and a marketing springboard for both a chemicals from biomass industry as well as potentially huge industry based on converting lignocellulosic materials to fuel ethanol. Such an industry may eventually arise because lignocellulosic materials are so inexpensive. While the cost and supply of grain severely limit its potential to replace a large percentage of gasoline, the situation for lignocellulose-derived ethanol is very different. Ample raw material supplies exist for lignocellulosic biomass. Further, because crop residues, grasses, hays and wood residues cost much less than grain, fuel ethanol produced from these lignocellulosic materials can potentially cost much less than grain ethanol. Assuming biomass processing technology at a similar stage of maturity as petroleum processing technology, it has been shown that the cost of fuel ethanol from lignocellulosics should be in the $0.50–$0.70 per gallon range, assuming biomass costing about $30.00 per ton delivered. Given low cost corn stover, another estimate projects ethanol costs at less than $0.50 per gallon for large scale plants, assuming best laboratory yields. Clearly, there is real potential for low cost fuel ethanol from cellulosic biomass if the processing technology for conversion of biomass can be made both efficient and inexpensive. Processing technology is the subject of the final section of this article.

IV. BIOPROCESSING OF BIOMASS A. Historical Lessons from the Chemical and Petroleum Processing Industries 1. Importance of Raw Material and Processing Costs for Commodities The chemical and petroleum industries grew together over the past century. These industries add value to raw materials by converting them to commodity and speciality products. Processing technologies of various kinds are used including chemical, thermal, physical and biological methods. By long experience, it has been found that the cost to produce commodities depends on two major factors: (1) the cost of the raw material and (2) the cost of the conversion process. The industries that produce chemicals and fuels from petroleum are characterized by high raw material costs relative to processing costs. Typically

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50–70% of the cost to produce a commodity product from petroleum is due to the petroleum cost itself. This is why gasoline prices fluctuate so widely when crude oil prices change. However, for the analogous biobased products industries, the processing costs predominate, rather than raw material costs. Therefore, a given percentage decrease in processing costs has much more impact on the profitability and economic competitiveness of biobased industrial products than does the same percentage decrease in raw material costs. As we have seen, the cost per ton of biomass raw materials is generally comparable to (e.g., corn grain) or much less (e.g., corn stover) than the cost of petroleum. Because of this fact, there is real potential for biobased products to be cost competitive with petroleum products if we can learn how to reduce the costs of processing biomass to desired products. Before discussing specific technical areas that seem to offer the best opportunities to reduce processing costs, a brief discussion of several lessons from the petroleum and chemical industries will be useful. 2. Need for Complete Raw Material Utilization This point is so elementary that it is often overlooked. For processes producing millions of pounds of biobased plastics or billions of gallons of fuel ethanol per year, essentially all of the raw material must be converted to saleable products, or at a minimum, not into wastes requiring expensive treatment and disposal. The petroleum refining industry has over time learned how to convert nearly all of the raw material into products. To compete effectively with this entrenched industry, the biobased products industry must become similarly efficient. Yield (conversion of raw material to products) must be increased and improved. A simple calculation will illustrate this point. If a raw material costing $0.10 per pound is converted into product at a yield of 0.9 pounds of product per pound of raw material, then the raw material cost is about $0.11 per pound of product. If the same raw material is converted to product at a yield of only 0.5 pounds of product per pound of raw material, the raw material cost is now $0.20 per pound of product, nearly double the previous case. The petroleum industry is characterized by high yields; the biobased products industry must strive to improve its yields also. While low yields are a definite economic handicap, they may be an even more severe environmental (and by consequence an economic) handicap. Whatever portion of the raw material is not converted to saleable products becomes waste instead. These wastes must be treated before disposal, if disposal is possible. Liquid wastes from biobased products will likely be characterized by relatively low

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toxicity, but by high oxygen demands if processed in conventional sewage treatment facilities. Solid wastes from biomass processing could occupy large volumes of landfill space, and would tend also produce high oxygen demand liquid effluents from landfills. The volume of landfill space that might be occupied by these products is remarkable, particularly for biobased fuels. For example, corn stover contains approximately 10–12% of protein and fat, in addition to the 70–75% of the plant material that is carbohydrate and that could be converted to fuel ethanol in a large fermentation facility. If no economic uses are found for the protein and fat or if economical recovery processes are not developed, these biomass components will have to be burned, landfilled, or treated as sewage and the resulting sludge disposed of. A hypothetical ethanol plant producing 100 million gallons of ethanol per year (less than 0.1% of current U.S. gasoline demand) from 1 million tons of corn stover will also produce at least 100,000 tons of protein and fats. Assuming a bulk density of about 50 lb per cubic foot, this much protein and fat will occupy a volume equivalent to the surface area of a football field stacked approximately 100 ft deep. Clearly there is strong economic incentive to find uses for all of the components of biomass. Therefore, as has occurred with the oil refining industry, “biorefineries” will tend to emerge. These biorefineries will be large, highly integrated processing plants that will yield numerous products and will attempt to sell a pound of product for every pound of raw material entering the plant. Prototype biorefineries already exist, including corn-wet and dry mills, soybean processing facilities and pulp and paper mills. The number and variety of these biobased products will increase over time, as has occurred with the oil refining industry. Many biorefinery products can also be produced by oil refineries; including liquid fuels, organic chemicals and materials. However, biorefineries can make many other products that oil refineries cannot, including foods, feeds, and biochemicals. These additional capabilities give biorefineries a potential competitive edge and may provide increased financial stability. 3. Incremental Process Improvement As we have seen, the number of products from a refinery tends to increase with time. In addition, the processing technologies used by refineries tend to improve incrementally over time. This is partly due to research that improves or replaces existing processes, supported within the cost structure of a successful industry. Research targets those process elements that most impact the overall cost of conversion. Incremental cost reduction is also partly due to organizational learning that can only occur in a functioning

industry. The more biorefineries that are established and become successful, the more that will be built as risks decline and “know how” increases. The cumulative effect of incremental process improvement is to cause the raw material costs to eventually become the dominant cost factor. This has already occurred with the oil refining industry and will take place in the biomass processing industries as these are established and grow to maturity. In this regard, biorefineries have a significant potential advantage over petroleum refineries because plant-based raw materials are abundant, widely available and inexpensive. The availability and prices of plant raw materials may thus be more stable and predictable than those of petroleum. As we have seen, plant raw material prices are already comparable on a cost per ton basis with petroleum and coal. Over time, petroleum prices must rise to reflect the fact that it is a nonrenewable resource, while there is the potential to keep biomass costs low indefinitely. 4. Innovation and Risk While biorefineries have some inherent advantages over petroleum refineries, they also have some significant disadvantages. First, the costs and risks of petroleum refining are well understood and the commodity organic chemicals industry based on petroleum is very well developed. However, the costs and risks of biomass refining are not nearly so well understood, particularly for large scale plants converting lignocellulosic biomass into fuel ethanol. Innovation is always regarded as risky compared to the status quo. Investors demand a greater return on investment to compensate for these increased risks and they also require additional capital investment in the processing plant to reduce processing risks or uncertainties. Second, when the petroleum industry was new there was little or no competition for many of its products as they emerged. However, most of the potential new biobased products must compete with a comparable petroleum-derived product. These two factors are significant hurdles for biomass processing industries to overcome. A better fundamental understanding of the underlying science and technology for biomass conversion processes could reduce the risks of such processes as they are scaled up to commercial practice. Better fundamental understanding would reassure investors and allow them to reduce the return on investment required. Better fundamental understanding of the processes would also tend to reduce the processing equipment redundancy required because of lack of certainty about its proper functioning under commercial practice. Some of the key areas of biomass processing in which greater fundamental understanding is required are discussed below.

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154 B. Current Status of Biomass Processing Biomass processing to industrial products based on starch, sugar, and oilseed raw materials is partially developed. Fiber crop processing to pulp and paper is very well developed and is not discussed further here. Processing of lignocellulosic materials to industrial products other than pulp and paper is very limited. For processing of starch, sugar, and oilseed crops, a primary need is to develop additional industrial products from these raw materials. This is because a processing infrastructure, or at least the beginnings of one, already exists or could be developed for each of these raw materials. Therefore the capital risk of a totally new plant is not required. Corn wet and dry mills, sugar refineries for cane and beet sugar, and oilseed crushing mills already exist and industrial products are already produced from these raw materials. As additional products are developed and appear profitable, they can often be added to existing product lines at existing facilities, for instance in a corn wet mill. Corn wet millers have steadily increased the number and variety of their products over the past two decades or so. This trend is likely to continue. Growth of new biobased products at oilseed crushing mills appears to have been much slower. However, when circumstances appear favorable, totally new processing facilities can and will be built around specific new industrial products, as evidenced by the new plants for polylactic acid production announced and/or under construction. Many of these starch or sugar-based products might also be produced at even lower cost from inexpensive sugars generated in a lignocellulose conversion plant. Processing plants based on lignocellulose are struggling to become established, at least partly because the processing technology is underdeveloped and therefore relatively expensive compared to petroleum processing technology. C. Priorities for Developing Lignocellulose Biorefineries 1. Enhancing Yield and Feedstock Modification The emphasis in this article on development of processing technologies for large scale refining of lignocellulosic materials to industrial products is not intended to detract from at least two other important development areas that are not directly connected with processing technology. The first of these is yield. The profitability of biomass conversion industries using dedicated (grown for that purpose) feedstocks will be strongly affected by the yield of raw material (tons of dry lignocellulosic material produced per year per acre of land). Agronomic research to increase yields and develop improved plant varieties for biomass production

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is important and requires continuing attention. A key area of agronomic research, particularly for large-scale energy crops, is to reduce the amounts of fertilizers, pesticides, herbicides and other inputs required, both to minimize costs and to reduce potential environmental hazards. The second important area that is not directly process development is plant feedstock modification, either by breeding or genetic modification. As mentioned, feedstocks can be altered to make them easier to process, a major advantage of biomass feedstocks compared with petroleum raw materials. Feedstocks can also be altered to contain larger amounts of desirable components or even to contain entirely new components such as bioplastics. Careful integration of processing technology development and product recovery with feedstock modification is required to achieve the maximum benefits of these genetic manipulations. Presumably yields might also be affected by some of these modifications. Thus feedstock modification and yield enhancement should proceed as an integrated whole. 2. New Technologies Needed for Low Cost Lignocellulose Conversion While it is relatively easy to convert starchy materials such as corn to fermentable sugars and then to a variety of fermentation products, lignocellulosic materials are much more difficult to convert to fermentable sugars. The potential benefit of economical conversion of lignocellulosics to fermentable sugars is the much larger possible volumes and therefore lower costs of lignocellulose-derived sugars compared to starch-derived sugars. Such low cost sugars are a prerequisite to inexpensive fuel ethanol. Inexpensive sugars could also significantly reduce the costs of other biobased products such as polylactic acid or 1,3 propanediol. Three primary areas for new technology development are required to reduce the cost of producing fuel ethanol and commodity chemicals from lignocellulosic materials: (1) an effective and economical pretreatment to unlock the potentially fermentable sugars in lignocellulosic biomass or alternative processes that enable more biomass carbon to be converted to ethanol or other desired products, (2) inexpensive enzymes (called “cellulases”) to convert the sugar polymers in lignocellulose to fermentable sugars, and (3) microbes that can rapidly and completely convert the variety of five and six carbon sugars in lignocellulose to ethanol and other oxygenated chemicals. Several lignocellulose pretreatment processes have recently been developed that promise to be technically effective and affordable, and some of them are undergoing large scale testing and development. Advanced lignocellulose treatments include processing with ammonia and

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other bases, pressurized hot water and catalytic amounts of acid. Such pretreatments may eventually make it possible to convert a large array of lignocellulose residues into useful products. It has long been recognized that a technologically and economically successful lignocellulose pretreatment will not only unlock the sugars in plant material for conversion to industrial products but will also make these sugars more available for animal feeding. Considerable progress has been made in developing genetically engineered microorganisms that can utilize the complete range of sugars in lingocellulosic materials. Both genetically engineered bacteria and yeasts are now available that utilize both five and six carbon sugars and convert them to ethanol. However, less progress is apparent in production of low cost cellulase enzymes, an active area of development at this time. Fortunately, while research on cellulases will take both time and money, the research pathways required to achieve success, (i.e., much lower cost active cellulases), are relatively clear. It may also be possible to largely bypass the processes of cellulose pretreatment and enzymatic hydrolysis to produce fermentable sugars. One such approach is to gasify biomass to mixtures of carbon dioxide, hydrogen and carbon monoxide and then to ferment the resulting gas mixture to ethanol or other products. This amounts to a technological “end run” around the processes of biomass pretreatment and enzymatic hydrolysis, and could also significantly simplify the fermentation process. Two major technical obstacles to this approach include clean up of the gas stream, particularly to remove sulfur and nitrogencontaining compounds, and also the difficulty of transferring slightly soluble gases into a liquid fermentation mixture. 3. Generic Biomass Processing Technologies The following comments apply generally to biomass conversion, not just to lignocellulose conversion. Processing technologies that utilize microbes and enzymes have great potential for low cost biomass processing. Unlike most thermal and chemical processes, bioprocesses take place under relatively mild conditions of temperature and pressure. Higher temperatures and pressures add significantly to the cost of processing in conventional chemical industries so that advanced bioprocessing technologies have the potential to be less expensive than their non biological counterparts. Some advanced bioprocessing technologies utilizing microbes and enzymes have already been developed, for example, immobilized cell technology and simultaneous hydrolysis and fermentation of sugars from lignocellulosics. Bioprocesses result in stereospecific conversions (the particular arrangement of atoms in space) and produce relatively nontoxic byproducts. However, the

volume of such byproducts can be very large and there is a pressing need to find markets for all such products and byproducts. Bioprocessing research should therefore focus on (1) increasing processing rates to reduce the capital investment required, (2) increasing product yields to decrease the raw material costs and to reduce the load on the separation and waste disposal systems, and (3) increasing product concentrations to reduce separation costs. One drawback is that bioprocesses typically yield dilute aqueous product streams that require further processing to separate and purify products. Separation technologies for biobased products are typically less developed than separation technologies for comparable petroleum-based products. A major need is to find low cost ways of removing the large amounts of water that often accompany biobased products. In general, research on the underlying production processes should focus on the science and engineering required to overcome the most significant cost barriers to commercializing biobased products. Experience with commercial amino acid production illustrates the potential of combining inexpensive raw materials with advanced processing technologies. International amino acid markets were completely dominated by Japanese firms in the early 1980s. However, in the 1990s U.S. companies used inexpensive corn-based sugars and an advanced processing method, immobilized cell technology, to penetrate these markets and now occupy a significant part of the global amino acid trade. One of the reasons these corn-based sugars for amino acid production were so inexpensive is because they were produced in large, integrated biorefineries. The Archer Daniels Midland plant in Decatur, Illinois, is a prototypical biorefinery. At that location, a large corn wet-milling plant and a steam and electricity cogeneration station burning waste tires form the nucleus for several other plants that are highly integrated. These other plants are an ethanol facility as well as an amino acid production plant. Biorefineries, whether based on corn, straw or any other material, must aspire to a similar degree of integration and effectiveness in raw material conversion.

V. POTENTIAL AND LIMITATIONS OF BIOMASS AND BIOBASED INDUSTRIAL PRODUCTS A. Potential Benefits Biomass production and processing have the potential to give us a uniquely sustainable source of organic chemicals, organic materials (such as biopolymers) and liquid transportation fuels. Biomass can also help us sustainably

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156 produce electricity, although there are several other sustainable sources of electricity. Biomass is also uniquely a source of human food and animal feed. Because biomass production is widely dispersed geographically, biobased industrial products can potentially form the basis of both local and worldwide economic systems that are much more equitable and balanced. Also because biomass production is widely dispersed, resourcedriven international conflicts over petroleum might be minimized or avoided entirely. Biomass production is a key part of global cycles of carbon, oxygen, nitrogen, water and other compounds. If we intelligently produce and use biobased industrial products we may actually improve environmental quality and increase or enhance the stocks of “natural capital” such as soil, water and air upon which all life depends. Numerous opportunities also exist to integrate biomass production and processing with waste utilization and recovery of damaged or less fertile lands. For example, the organic fraction of municipal solid wastes might be combined with human and animal wastes and composted to enrich marginal soils producing perennial grasses for a bioethanol facility. Furthermore, since plants fix atmospheric carbon both in their above and below ground parts, the potential exists to continue to use petroleum and other fossil fuels indefinitely, balancing the amount of atmospheric carbon dioxide liberated by fossil fuel combustion with the uptake and fixation of carbon dioxide by plants. B. Potential Limitations of Biomass and Biobased Industrial Products Perhaps the most serious potential limitation of biomass and biobased industrial products is the possible conflict with food and feed production on the same land. While biomass utilization for organic chemicals and materials, done properly, is not likely to result in conflicts with food production, biomass production and utilization for liquid fuels such as ethanol might indeed conflict with food production. This is particularly true if fuel use efficiency does not increase dramatically over the time frame that biofuels are implemented. Food production will always be a higher human priority than production of fuels or plastics. This issue must be carefully considered and appropriate resolutions achieved if biobased industrial products, including biomass-derived liquid transportation fuels, are to provide us their full social, economic and environmental benefits. Some threats to biodiversity and water and soil quality are also possible from greatly increased levels of biobased industrial products. Erosion and increased contamination of soil and water with fertilizers, pesticides and herbicides might result from intensive production of biomass for biobased products. Disposal of biobased

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products, done with reasonable care, should not have environmental impacts more severe than the corresponding petroleum-derived products. In fact, biobased products are well suited to composting or other resource recovery approaches that return their constituent atoms to the global cycles of materials. C. Achieving the Benefits of Biobased Products While the potential benefits of biobased products are certainly real, so are their limitations and possible problems. One way of achieving the benefits of biomass processing to biobased products is to do careful, system-level studies of specific products in order to anticipate and resolve potential problems before large industries are launched and the damage is done. Life cycle analysis is suited to such system studies. For example, there is an obvious potential for biomass production for biobased products to conflict with food production. Careful studies are required to anticipate and resolve such conflicts before they occur. One potential resolution of this apparent conflict with food and fuel production is to coproduce foods and animal feeds with fuel and chemical production from biomass. Most biomass produced is actually fed to animals, rather than directly to humans. Since most biomass also contains protein (required in all animal diets), the potential exists to recover this biomass protein in a biorefinery and use it to feed animals, or perhaps even people. Assuming an average protein content of 10% in grasses and residues, and assuming 80% recovery of this protein in biorefineries also producing ethanol fuel, about 1 billion tons of grass would be required to replace all of the protein currently produced worldwide as high protein meals from oilseeds such as soybeans. The equivalent amount of ethanol produced would be about 100 billion gallons per year, about half of the U.S. demand for liquid transportation fuels. Similarly, the calories (food energy) in lignocellulosic materials are not very available for animal digestion and they are essentially useless in human nutrition. However, if the technical roadblock of lignocellulose pretreatment for production of fuels is resolved, it will also be resolved for pretreatment to increase the food and feed calories available from lignocellulosics. For example, ruminant animals typically digest less than half of the calories potentially available in grasses. If pretreatments make those calories 90% percent available both for fermentation to ethanol and also for animal feeding, then the treatment of about 4 billion tons per year of grasses will make available for feed (or food) and fuel uses new, additional calories approximately equal to the calories contained in the entire world grain crop of about two billion tons per year. Thus while both

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the protein and calorie issues in biomass processing need careful analysis, it may be possible to actually increase, rather than decrease, world food and feed resources as a consequence of large-scale biomass processing to fuels. While the potential difficulties of biomass processing are certainly real, so are the potential benefits. In essence, we have the unique opportunity to guide and develop a new industry so that it improves the quality of our environment at the same time it provides many economic, environmental and social benefits. Both the opportunity and the challenge are ours.

SEE ALSO THE FOLLOWING ARTICLES BIOENERGETICS • BIOMASS UTILIZATION, LIMITS OF • BIOREACTORS • ENERGY EFFICIENCY COMPARISONS BETWEEN COUNTRIES • ENERGY FLOWS IN ECOLOGY AND IN THE ECONOMY • ENERGY RESOURCES AND RESERVES • WASTE-TO-ENERGY SYSTEMS

BIBLIOGRAPHY Hawken, P., Lovins, A., and Hunter Lovins, L (1999). “Natural Capitalism,” Little, Brown and Company, Boston. Klass, D. L. (1995). “Fuels from Biomass,” In “Encyclopedia of Energy Technology and the Environment,” (A. Bisio and S. Boots, eds.), Vol. 2, pp. 1445–1496, Wiley, New York. Lugar, R. G., and Woolsey, R. J. (1999). “The new petroleum,”Foreign Affairs 78(1), 88–102. Lynd, L. R. (1996). “Overview and evaluation of fuel ethanol from cellulosic biomass: Technology, economics, the environment and policy,” Annu. Rev. Energy Environment 21, 403–465. Lynd, L. R., Wyman, C. A., and Gerngross, T. U. (1999). “Biocommodity engineering,” Biotechnology Progr. 15(5), 777–793. McLaren, J., and Faulkner, D. (1999). “The Technology Roadmap for Plant/Crop-Based Renewable Resources 2020,” U.S. Department of Energy, Washington, DC, DOE/GO-10099-706. Morris, D., and Ahmed, I. (1992). “The Carbohydrate Economy: Making Chemicals and Industrial Materials from Plant Matter,” Institute for Local Self-Reliance, Washington, DC. National Research Council (2000). “Biobased Industrial Products: Priorities for Research and Commercialization,” National Academy Press, Washington, DC.

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Biomaterials, Synthetic Synthesis, Fabrication, and Applications Carole C. Perry The Nottingham Trent University

I. Introduction to Medical Biomaterials II. Aspects of the Structural Chemistry of Natural Materials Used in the Human Body III. General Repair Mechanisms and Biocompatibility IV. Materials of Construction V. The Way Forward, Tissue Engineering

GLOSSARY Biomaterials A substance that is used in prostheses or in medical devices designed for contact with the living body for an intended application and for a planned time period. Biocompatibility The capability of being harmonious with biological life without causing toxic or injurious effect. Ceramics Stiff, brittle materials that are generally prepared by high temperature methods; the resulting materials are insoluble in water. Composites Materials composed of a mixture or combination of two or more microconstituents or macroconstituents that differ in form and chemical composition and that are essentially insoluble in each other. Metals Any of a class of elements that generally are solid at ordinary temperature, have a grayish color and a

shiny surface, and will conduct heat and electricity well. Polymers Large molecules formed by the union of at least five identical monomers. Tissue engineering The study of cellular responses to materials implants, manipulation of the healing environment to control the structure of regenerated tissue, the production of cells and tissues for transplantation into the body, and the development of a quantitative understanding of biological energetics.

I. INTRODUCTION TO MEDICAL BIOMATERIALS The normal description of a biomaterial is “a substance that is used in prostheses or in medical devices designed for contact with the living body for an intended application and for a planned time period.” The development of

173

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biomaterials has occurred in response to the growing number of patients afflicted with traumatic and nontraumatic conditions. As the population grows older there is an increased need for medical devices to replace damaged or worn tissues. The market is a billion dollar per year market and requires the skills of clinicians, surgeons, engineers, chemists, physicists and materials scientists to work cooperatively in the development of materials for clinical use. There are five classes of biomaterials; metals, ceramics, biological materials/polymers, synthetic polymers, and composites. The choice of material to replace biological tissue is largely governed by the physical properties of both the natural tissue and the proposed replacement. In general, natural and synthetic polymers are used to replace skin, tendon, ligament, breast, eye, vascular systems, and facial tissues, and metals, ceramics and composites are used to replace or reinforce bone and dentin. Replacements for these natural tissues clearly require materials of different strength. Table I shows the strength of the main groups of natural materials together with the synthetic counterparts used in the development of replacement materials. It is often the case that the strengths of the materials used to replace natural components are stronger and/or stiffer which often leads to problems of compatibility both in respect of mechanical behaviour of the implant within the host and in terms of the biologic response. TABLE I Physical Properties of Tissues and Materials Used in Their Replacement Material Natural materials Soft tissue Arterial wall Hyaline cartilage Skin Tendon/ligament Hard tissue (bone) Cortical Cancellous (porous) Synthetic materials Polymers Synthetic rubber Glassy Crystalline Metal alloys Steel Cobalt Platinum Titanium Ceramics Oxides Hydroxylapatite Composites Fibers Matrices

Ultimate strength (Mpa)

Modulus (MPa)

0.5–1.72 1.3–1.8 2.5–16 30–300

1.0 0.4–19 6–40 65–2500

30–211 51–193

16–20 (GPa) 4.6–15 (GPa)

10–12 25–100 22–40

4 1.6–2.6 (GPa) (0.015–1) (GPa)

480–655 655–1400 152–485 550–680

193 (Gpa) 195 (GPa) 147 (GPa) 100–105 (GPa)

90–380 (Gpa) 600

160–4000 (GPa) 19 (GPa)

0.9–4.5 (Gpa) 41–106

62–577 (GPa) 0.3–3.1

Many of the materials used today in the clinical environment were not originally engineered for biomaterials applications. In crude terms, they became “biomaterials” when, by a series of trial-and-error experimentation, they were implanted in the human body in a variety of forms and found to “work.” Clearly there were many other materials which did not ‘work’ causing at the least perhaps pain and discomfort to patients and at the worse unnecessary suffering and death. Increase in litigation for problems allegedly caused by biomaterials has caused some companies to remove products from sale in this area and may lead to a distinct shortage of available materials for device fabrication. For a variety of reasons, trial-and-error optimization is not the way forward for the production of the next generation of materials to be used in the human body. What is required is the systematic design of a wide range of materials with specific functions for predefined medical use. The goal is to produce biomaterials that smoothly integrate into living systems rather than fighting against the normal functioning of a living body. There are many factors to consider in order to understand how this might be accomplished. The factors include the structure of the original material to be replaced or augmented, its physiology, anatomy, biochemistry and biomechanics including pathophysiological changes that have necessitated the use of a substitute biomaterial. In addition, as devices are often present in the body for considerable periods of time then it is necessary to understand the natural degenerative processes of normal tissues, particularly in relation to the biomaterial substitute. This latter area is at present very poorly understood. All of the above clearly impact on the design and development of materials for clinical usage. Thus materials need to be developed with a clear understanding of the nature and extent of interactions between the device (whatever it is) and the surrounding tissue. It cannot be emphasised too strongly the importance of biocompatibility in the development of the next generation of materials for applications in a biological environment. This chapter will describe the materials currently used as biomaterials and routes to their formation. It will describe some aspects of the structural chemistry of natural materials that are to be replaced or augmented and it will look at the way forward for the design of materials for use in the medical environment in the 21st century.

II. ASPECTS OF THE STRUCTURAL CHEMISTRY OF NATURAL MATERIALS USED IN THE HUMAN BODY Biological organisms make use of proteins, polysaccharides and combinations of these two types of molecule in the polymeric phases that are found in a living organism together with simple calcium salts. Chemical composition

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and structure both play important roles in determining the properties of the natural polymeric phase whether it is used alone or in a composite form. A. Natural Polymers The key to the mechanical properties of fibrous materials (collagen, silks, chitin and cellulose are all natural examples and polyethylene and nylon are examples of synthetic polymers) lies in their structure, in particular the extent of their crystallinity. Crystallinity equates with material rigidity, hardness and toughness, Table I. For crystalline polymers to form they must have great regularity of chain structure and be linear in form. The presence of more than one stereo-isomeric form of the polymer can cause problems in structural alignment for synthetic polymers but this is not a problem in natural polymers. Collagen or cellulose as only one stereo-isomer is utilized in the building of these structures. When the level of crystallinity is less than 20%, the crystalline regions act as cross-links in an amorphous polymer network with materials being mechanically similar to cross-linked rubbers. Above 40% crystallinity the materials become quite rigid and the mechanical properties of the polymers are largely timeindependent. The behavior of drawn fibers, such as those produced from synthetic polymers may be further different from the bulk material as the regions of amorphous character are greatly extended and aligned perpendicular to the stress direction. In general, the Young’s modulus (the stress–strain curve) of a bulk crystallized polymer is two to three orders of magnitude greater than that of an amorphous polymer such as rubber and the modulus of oriented, crystalline fibers is another order of magnitude greater still. Collagen is a prime example of an important crystalline polymer in mammals and is found in both rigid (e.g., bone) and pliant materials (e.g., tendon, skin and cartilage) (Table II). Each collagen chain is ca. 1000 amino acids in length and is dominated by a 338 contiguous repeating triplet sequence in which every third amino acid is glycine. Proline and hydroxyproline together account for about 20% of the total amino acids. The structure of collagen is based on the helical arrangement of three noncoaxial, helical polypeptides, stabilized by inter-chain and intra-chain hydrogen bonds with all the glycines facing the center of the triple helix. Individual collagen molecules are ca. 280 nm in length and 1.5 nm in width with adjacent molecules being transposed in position relative to one another by one quarter of their length in the axial direction. Collagen is the protein used in tissues where strength or toughness is required. In addition to the effect of its intrinsic chemical structure on the observed mechanical properties, part of its toughness arises from specific cross-links

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175 between the molecules. These naturally increase with age leading to a less flexible material as someone gets older. Elastin is used in situations where a highly elastic fiber is needed such as in ligaments and blood vessels (Table II). The polypeptide chain of elastin contains high levels of both glycine and alanine, is flexible and easily extended. To prevent infinite extension the peptide sequence contains frequent lysine side chains that can be involved in crosslinks that allow the fibers to “snap-back” on removal of tension. Elastin is only ever found as fine fibers and is usually found in association with collagen and glycosaminoglycans. The hydrophobic character of the protein (95% of the amino acids have nonpolar side chains) leads to water becoming more ordered and the polymer less ordered when fibers of the material are stretched. Carbohydrate-based natural polymers include glycosaminoglycans (polysaccharides containing amino acids formerly known as mucopolysaccharides), proteoglycans (covalently bonded complexes of protein and glycosaminoglycan in which the polysaccharide is the dominant feature) and protein–polysaccharide complexes where a complex is held together through noncovalent linkages. Important examples are the chondroitin sulfates and keratan sulfates of connective tissue, the dermatan sulfates of skin and hyaluronic acid. All are polymers of repeating disaccharide units in which one of the sugars is either N-acetylglucosamine or N-acetylgalactosamine. Examples are given in Table II. A major function of this group of molecules is the formation of a matrix to support and surround the protein components of skin and connective tissue. A schematic view of the proteoglycan complex in cartilage used to bind to collagen in a strong network is shown in Fig. 1. The filamentous structure contains at its heart a single molecule of hyaluronic acid to which is attached via noncovalent interactions proteins known as “core” proteins. These in turn have chondroitin sulfate and

FIGURE 1 A schematic view of the proteoglycan complex in cartilage used to bind to collagen in a strong network.

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Biomaterials, Synthetic Synthesis, Fabrication, and Applications TABLE II Natural polymer Collagen

Structural unit

Elastin

(gly-x-pro/Hpro)n where x is another amino acid. Triple helices are built up from the basic structural units and held together by inter- and intra-molecular bonds between helices to form fibers (gly-val-gly-val-pro)n

Chondroitin sulphate

Polypeptide rich in glycine and alanine, 95% hydrophobic residues Lysine side-chains used in cross-linking to give fibers Polymer of glucuronic acid and sulfated n-acetylglucosamine

Keratan sulphate

Polymer of galactose and sulfated n-acetylgalactosamine

Hyaluronic acid

Polymer of glucuronic acid and n-acetylglucosamine

keratan sulfate chains covalently bound to them. The properties of these molecules and their protein–polysaccharide complexes are largely determined by the poly-anionic character of the glycosaminoglycans. Both carboxyl and sulfated groups are ionized at physiological pH to give highly charged polymeric molecules where the molecule takes on a highly expanded form in solution. Perhaps unusally, glycosaminoglycan complexes of connective tissue may also contain a small amount of silicon (ca. 1 silicon atom per 100 sugar residues) as a cross-linking agent between adjacent chains. Hyaluronic acid has additional functions within the body due to its high solubility in water. In addition to the properties described above, molecules are able to interact with one another at low concentrations to form entanglement networks and produce solutions with viscoelastic properties. In addition, if some permanent cross-bridges can form then gel-like structures with rubber-elastic prop-

erties can result. Hyaluronic acid is thus able to act as a viscosity modifier and a lubricating agent in the synovial fluid of joints and in the vitreous humor of the eye. B. Natural Pliant Composites Pliant composites are tendon, skin, and cartilage, all of which contain fibers (in some proportion) and a chemical matrix to support and modify the behavior of the high strength material. Collagen is generally used as the fibrillar component with differences in the thickness of these fibers (15 to 150 nm) being related to the required mechanical properties of the composite. There are differences in the extent of cross-linking between the collagen molecules and in the nature and organization of the fibrils and the matrix in which it is found. Tendon is the structure that enables the rigid attachment of muscle to bone, and as such it must transmit the muscle

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force with a minimum of loss. This is achieved through the parallel arrangement of collagen fibers to form ropelike structures with a high modulus of elasticity and high tensile strength. Tendons, such as the Achilles tendon, that are under a lot of stress probably contain collagen fibers that are more highly cross-linked to reduce the rate of stress–relaxation to an insignificant level. Skin is a complex tissue made up of a thick collagenous layer (the dermis), a basement membrane and an overlying keratinized epidermal layer. The mechanical properties of skin arise principally from the dermis which is a three-dimensional feltwork of continuous collagen fibers embedded in a protein–polysaccharide matrix rich in both dermatan sulfate and hyaluronic acid, the latter being used to reduce frictional wear between the collagen fibers. Elastin fibers are distributed throughout the tissue or concentrated in the lower layers of the dermis depending on the precise location of the skin within the body. The arrangement of elastin fibers within a collagen framework results in a material showing rubber-elastic properties at small extensions but is limited at longer extensions by the dimensions of the collagen framework. Cartilage acts as a material that maintains the shape of ears, the nose, and the invertebral disc. Cartilage contains collagen fibers, a proteoglycan matrix phase rich in chondroitin 4- and 6-sulfate and sometimes elastin fibers and yet the material must be able to resist compression and bending forces. Cartilage can be thought of as a hydrostatic system in which the fluid element is provided by the hydration water of the proteoglycan gel and the container provided by the collagen fiber meshwork which immobilizes the molecules of this gel. Thus, the rigidity of the system arises from the osmotic swelling of the proteoglycan gel against the constraints imposed by the collagen fiber system. Cartilage may additionally be mineralized and will be discussed below in conjunction with other mineralized tissues. C. Natural Mineralized Tissues, Bone, Cartilage, and Enamel Vertebrates construct their skeletal and dental hard parts from calcium phosphates with calcium carbonates being used for balance organs and egg shells. Bone, dentin, enamel, and mineralized cartilage all contain crystalline calcium apatite phases but the crystals exhibit different sizes, compositions, and ultrastructural organization. Apart from enamel they all contain collagen fibers, and additional inorganic salts and biomolecules. Bone has unusual physical and mechanical properties in that it is able to support its own weight, withstand acute forces, bend without shattering and can be flexible without breaking within predefined limits. Bone also acts as

TABLE III Polymers Used in Medical Devices Polymer Polyethylene Polyethylene terphthalate Polymethylmethacrylate Polydimethylsiloxane Polyurethane

Medical device applications Hip, tendon/ligament implants and facial implants Aortic, tendon/ligament and facial implants Intraocular lens, contact lenses and bone cement Breast, facial and tendon implants Breast, vascular and skin implants

an ion reservoir for both cations and anions. In material terms bone is a three-phase material; the organic fibers (collagen) can be compared to steel cables in reinforced cement, the inorganic crystalline phase (carbonated hydroxyapatite) to a heat-treated ceramic and the bone matrix to a base substance which performs various cellular functions. The unique physical and mechanical properties of bone are a direct result of the atomic and molecular interactions intrinsic to this unusual composite material. Bone comprises collagen fibrils intimately associated in an orderly fashion with small calcium phosphate crystals. The crystals are of carbonated-hydroxyapatite more correctly described as the mineral dahllite. The formula Ca8.3 (PO4 )4.3 (CO3 )x (HPO4 )y (OH)0.3 represents bone mineral with the values of X and Y changing with age (Y decreases and X increases with increasing age, whereas X+Y remains constant with age equal to 1.7!). Traces of other elements such as silicon may also be associated with deposition of the mineral phase. The individual crystals have an average length of 50 nm (range 20–150 nm), width 25 nm (range 10–80 nm) and thickness of 2–5 nm. In addition to collagen at 85–90% of the detectable protein there are more than 200 noncollagenous proteins (NCPs) present. The three major classes of NCP’s are acidic glycoproteins, proteoglycans and Gla- (γ -carboxyglutamic acid) proteins. The acidic glycoproteins contain considerable amounts of the amino acids phosphoserine, phosphothreonine, and γ -carboxyglutamic acid. The phosphopoteins are intimately associated with the initiation and regulation of crystal growth and may serve as a source of inorganic phosphate on enzymatic release by phosphatases. The proteoglycans have one or more (negatively) charged glycosaminoglycan chains attached to the main protein chain and may be present to inhibit crystal growth due to their negative charge and to reserve the extracellular space for future calcium phosphate crystal growth due to their ability to structure water. Both these classes of proteins together with alkaline phosphatase are found in a range of mineralized tissues and their wide distribution suggests that they have a basic role to play in controlling

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178 mineralization systems. Bone Gla-containing proteins are unique to bone and dentin and as such are expected to have a specific functional role to fulfill in these tissues. Stages in the formation of bone are: (1) synthesis and extracellular assembly of the organic matrix framework, (2) mineralization of the framework, and (3) secondary mineralization as the bone constantly forms and reforms. All of the salts and biomolecules associated with bone described above will play their own role(s) in the development of bone, the structure of which can vary considerably according to the location and use to which the resulting natural composite is to be used. Figure 2 shows pictorially

Biomaterials, Synthetic Synthesis, Fabrication, and Applications

the difference in porosity of cortical and cancellous bone with the former being found where load bearing is important. Bone is constantly in a state of dynamic equilibrium with its environment and changes with age. Changes with time will also be expected for diseased states and when foreign bodies (e.g., implants) are in close proximity to these phases although much less is known for such situations. An understanding of the structure and dynamics of natural materials should enable to design of materials for their replacement which will be chemically more compatible with those they are seeking to replace. Mineralized cartilage contains much thinner fibers of collagen than are found in bone, high levels of water of hydration, and hydroxyapatite crystals, although there is no regular organization of the crystallites with respect to the collagen matrix. Enamel is formed via the assembly of a matrix comprising both collagenous and noncollagenous proteins into which large oriented hydroxyapatite crystals are formed. The crystals may be of the order of 100 microns in length, 0.05 microns in diameter, and with an hexagonal cross-section. At maturity water and protein (including collagen) are removed from the tooth leaving a collagenfree composite.

III. GENERAL REPAIR MECHANISMS AND BIOCOMPATIBILITY

FIGURE 2 Schematic drawings of (a) human corticalbone and (b) humand cancellous bone. Note the difference in packing density and porosity between the two idealized structures. (Reprinted with permission from Perry, C. C. (1998). In “Chemistry of Advanced Materials.” (L. V. Interrante and M. J. Hampden-Smith, eds.), pp. 499–562, Wiley VCH, New York.

Under normal circumstances most tissues in the body are able to repair themselves although the process and the presence or absence of scarring is tissue dependent. Bone repair occurs either through formation of membranous bone or through mineralization of cartilage. In facial bones, clavicle, mandible, and subperiosteal bones, membranous bone growth involves calcification of osteoid tissue (endochondral bone formation). In long bones the stages of repair include induction, inflammation, soft callus formation, callus calcification and remodeling. Cartilage and skin can also repair themselves although scarring does occur. For skin, repair involves inflammation, immunity, blood clotting, platelet aggregation, fibrinolysis, and activation of complement and kinin systems. In the absence of a chronic inflammatory response, dermal wounds are repaired through deposition and remodeling of collagen to form scar tissue. Enamel is not repaired by the body. Early studies on biomaterials were based upon the idea that implants would not degrade in the human body or be involved in biological reactions. Hence the search was for bioinert materials, whatever their chemical composition. However, no material implanted in living tissue is inert and all materials elicit a response from the host tissue. For

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SCHEME 1 Condensation polymerization.

toxic materials, the surrounding tissue dies. For nontoxic materials and those that dissolve, the surrounding tissue replaces the implant. For biologically nontoxic and inactive materials, a fibrous tissue capsule of variable thickness can form. For nontoxic and biologically active materials, an interfacial bond forms. Materials used in medical procedures should be biocompatible with the host tissue. Biocompatibility of an implant material is deemed optimal if it promotes the formation of normal tissue at its surface, and in addition, if a contiguous interface capable of supporting the loads that normally occur at the site of implantation is established. Hence the current goal is to produce materials that are recognized and assimilated by the body.

IV. MATERIALS OF CONSTRUCTION All medical implants are composed of polymers, metals, ceramics, or mixtures and composites of these materials. Tissue replacement with synthetic materials requires selection of a material or materials with physical properties most similar to those of the natural tissue (Table I). A. Synthetic Polymers These are the most widely used materials in health care and are used in almost all phases of medical and/or dental treatment. Typical polymers in use are listed in Table II together with selected uses. 1. Synthesis Polymers are large molecules made from many smaller units called monomers that are chemically bonded to form

the polymer. If only one species of monomer is used to build a macromolecule the product is termed an homopolymer, normally referred to as a polymer. If two types of monomer unit are used the material is known as a copolymer and if three different monomers are used then a terpolymer results. Polymers may be formed by condensation reactions between complimentary functional groups to make poly(esters), poly(amides) and poly(urethanes) (Scheme 1). They may also be formed by free radical polymerisation of unsaturated compounds to give addition polymers (Scheme 2). Examples of this class include poly(ethylene), poly(vinyl chloride) and poly (methyl methacrylate). A third route to polymers includes ring opening reactions as in the formation of nylon-6 from ε-caprolactam (Scheme 3). The structure, chemical functionality and physical properties of the polymer phase and hence its potential in medical devices depends on the monomers used and the method of synthesis. The presence of more than one functional monomer can lead to different arrangements of the monomers in the polymer chain (Fig. 3). These structural variations, including effects due to chirality have an effect on the way in which the polymer chains can pack together and hence they have an effect on the physical properties of the material itself. Polymers are often compounded with a range of other materials to lower the cost and improve the performance of the proposed polymer device. Accelerators are used to increase the extent and rate at which cross-linking between the chains occurs. Antioxidants such as amines and hydroquinones are added to minimize the cracking of a device when exposed to oxidants. Fillers such as carbon black,

SCHEME 2 Addition polymerization.

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SCHEME 3 Ring opening polymerization.

glass fiber, mica, and silica can be used to reinforce polymers and improve their mechanical properties. There are problems associated with the use of many of these additives because of their low molecular weight (in relation to the polymer phase) as they may be leached from the device causing deleterious effects on the device itself and on the human body. Not only can there be problems due to leaching of additives but also there may be problems due to the very components used in the synthesis of a material. For example, contaminants found in silicone polymers due to the synthesis route include; siloxane monomers, platinum catalysts, and peroxide derivatives. This problem is also significant in the production of bioceramics and composites for use in biomedical applications. The mechanical properties of polymers are a consequence of the distribution and packing of the polymer chains (degree of crystallinity) and the transition temperature at which they change from a viscoelastic material to a rigid glass. They cover a wide range of strengths (as measured by values for the Young’s modulus) and can therefore be used for a wide range of applications.

2. Polymer Modification Polymers are required for use under aqueous conditions where the binding of proteins (for recognition by the body) and cells is required in order to produce a favourable biological response. Polystyrene, polyethylene and terphthalate, polyfluoroethylene and perfluorinated ethylene propylene copolymer are poor supports and require postsynthesis modification of their surfaces to improve biocompatibility. Gas plasma (glow discharge) methods have become popular as a way of fabricating a range of surface chemistries. Surfaces are produced with functionalities rich in oxygen groups including O, OH, surface sulfonate and carboxyl which are, in principle, more compatible with biological tissues than the carbon–carbon and carbon–hydrogen bonds present in the parent polymers. It is also possible to use grafting (long used in industries based on polymers) to modify the surface chemistry of polymers. A grafted surface can be produced primaril by graft polymerization (often a free radical process) of monomers or the covalent coupling of existing

FIGURE 3 Schematic of polymer structures possible when two different monomers are used in the synthesis.

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polymer molecules onto the substrate polymer surface. A terminal group reactive to functional groups present on the substrate polymer surface is required for the polymer chains to be used in the coupling reaction, while the graft polymerization method needs active species on the substrate polymer to initiate radical polymerization. An apparently grafted surface can also be produced by physical adsorption of existing polymers. The adsorbed chains are readily desorbed upon immersion in solvents or washing with surfactants unless the adsorptive force is remarkably strong. Polyethyleneoxide (PEO) has been used to modify artificial surfaces by the above chemical surface modification methods with the establishment of PEO-surface and PEO-interpenetrating networks. An alternative simple and cost-effective approach is to use the melt blend technique where small amounts of protein compatible additives, such as PEO, polyvinyl alcohol (PVA), poly(ethyl oxazoline) (PEOX), and poly(vinyl pyrrolidine) (PNVP) have been used to modify the substrate polymer. The base polymer could be a single polymer or mixture of ethylene–vinyl acetate (EVA), polypropylene (PP), glycol modified poly(ethylene terephthalate) (PETG), poly(methylmethacrylate) (PMMA), styrene– butadiene copolymer and polyamide-based copolymers. Materials modified by these techniques can show enhanced protein adhesion important for general biocompatibility or show reduced protein adsorption which is useful in the production of blood-contacting devices, chromatographic supports, coatings to minimize biofouling, separation membranes, contact lenses, immunoassays, protein drug-contacting materials, etc. 3. Biodegradable Polymers Although there are many medical applications for polymers which require materials to be stable over a long time period, there are also devices such as sutures, small bone fixation devices, skin grafts, and drug delivery systems where polymers that break down in a controlled fashion are required. Materials that can be tailored to break down either unaided or by enzyme-assisted pathways under the conditions inherent in the location where the implant is found are desired. Two main factors affect the biodegradability of polymers. The first is the chemical composition of the material and the presence of hydrolysable and/or oxidizable chemical groups in the main chain, suitable substituents in side chains, stereoconfiguration, balance of hydrophilicity and hydrophobicity, and conformational flexibility all contribute to the biodegradability of synthetic polymers. The second aspect is the morphology of the polymer sample with amorphous polymer regions degrading prior to crystalline and cross-linked regions. Functional groups such

as amides, enamines, enol-ketones, esters, ureas, and urethanes when present in polymers all show some biodegradability. Materials being developed include lactide/glycolide polymers, polyorthoesters, derivatives of pseudoand poly-amino acids such as poly(l-glutamate), polyphosphazenes, poly(∈-caprolactone) and tyrosine– polycarbonates. Advantages of these materials lies in their ease of processability and their biocompatibility with extracellular matrix components present in the body. They may be made as high surface area, porous devices thereby allowing cell movement and adhesion within the device both important for the assimilation of the material within the body and in the eventual replacement of the biodegradable component with the bodies own tissue. Future applications of degradable polymers may involve their use in a foam form as scaffolds for the regeneration of specific tissues such as liver, bone, cartilage, or vascular walls. B. Polymers from Natural Sources In the search for biocompatible materials scientists have taken naturally occurring polymers and modifed the structure of the materials for a range of end uses. Materials based on collagen and hyaluronic acid are in common use for both dental, ophthalmological and maxillofacial reconstructive work where the “natural” polymer phase is used to fill out defects in the bone thereby providing a matrix around which bone will develop. Collagen is used in film, membrane, solution, gel, and sponge forms. It is also used in conjunction with glucosaminoglycans, tricalcium phosphate, hydroxyapatite, allogeneic bone, cells, and with drugs such as tetracycline. Most implant procedures where collagen has been used have required the implant to be a permanent feature in the human body. The “collagen” used was usually partially degraded, enzyme-extracted collagen, or had been stabilized by cross-linking it with cytotoxic glutaraldehyde or chromium salts, or else had been assembled into nonnatural polymeric structures such as films and sponges. An alternative approach is to maintain as much of the biological and organizational structure of collagen as possible using continuous collagen threads to make collagen fibers which can then be used to knit and weave fabrics with a structure more akin to that found naturally. Many different materials have been fabricated each with different bulk and extensibility properties. Materials derived from hyaluronic acid are conventionally esterified (either 100% or fractional amounts) and the acid esters so produced have physicochemical properties which are significantly different from those of the parent molecule. Ethylhyaluronic acid ester (HYAFF-07)

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TABLE IV Metals Used in Medical Devices Metal

Medical device applications

Cobalt–chromium alloys

Dental applicances, fracture plates, heart valves, joint components, nails, screws Conductive leads, joint components, pacemaker cases, nails, screws Fracture plates

Titanium alloys Stainless steel

and benzyl hyaluronic acid ester (HYAFF-11) are used to replace short nerve segments and in wound healing. The materials can be used as threads, films, fabrics and sponges and additional aplications are expected in plastic surgery and orthopedics. C. Metals Metals have a large range of applications as devices for fracture fixation, joint replacement, surgical instruments, external splints, braces and traction apparatus, as well as dental amalgams (Table IV). The high modulus and yield point coupled with the ductility of metals makes them suitable for bearing heavy loads without large deformations and permanent size changes. Metals are generally inert and if the composition is chosen carefully do not degrade in a saline environment. Metals are crystalline materials with a specific arrangement of metal atoms within a crystal lattice. Figure 4 shows the arrangements of atoms in the common crystal packing arrangements adopted by metals. The limits of a perfect crystal lattice are defined by grain boundaries where individual perfect crystals come together (Fig. 5). It is possible to

FIGURE 4 Common crystal packing arrangements for metals.

FIGURE 5 Schematic diagram of grain boundaries between crystallites. The boundaries may occupy only one row of atoms or more.

incorporate a wide range of different atoms either within the crystal lattice, at interstitial sites within the crystal lattice (spaces/sites where atoms or ions are not normally located), or at grain boundaries and thus a multitude of metal-containing materials can be made. The deformation characteristics of a metal are determined by the grain size of the crystals as imperfections are concentrated at the grain boundaries. Mixing of metal atoms of different sizes as in the production of an alloy can serve to modify the properties of the metallic phase. Metallic elements used in the formation of implants include: aluminium (Al), cobalt (Co), chromium (Cr), copper (Cu), gold (Au), iridium (Ir), iron (Fe), manganese (Mn), molybdenum (Mo), nickel (Ni), niobium (Nb), palladium (Pd), platinum (Pt), silver (Ag), tantalum (Ta), tin (Sn), titanium (Ti), vanadium (V), tungsten (W), zinc (Zn) and zirconium (Zr). Nonmetallic elements that are used to modify the properties of the metallic phases include carbon (C), nitrogen (N), phosphorous (P), sulfur (S), and silicon (Si). The metals and alloys that were originally used in the medical environment were selected on the basis of their strength and ductility although their original genesis may have been for a totally different purpose. To surmount problems due to corrosion under saline conditions alloys (homogeneous mixtures of the metallic elements at the atomic level) have been developed to render materials passive to corrosion. Another method is to promote the formation of an adherent oxide layer, often by acid treatment of the metallic phase. Both methods are currently used. Although the properties of metals can be modified by the chemical addition of different atoms to the alloy mixture, physical treatments such as annealing, precipitation hardening, tempering, work hardening, and polishing can also modify the modulus, toughness, and surface properties of the metallic phase. Processing of the metallic material is necessary to produce functional components and a combination of brazing (complex part formed by heating in the presence of another metallic material), drawing

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(wires and sheets formed from metallic ingots), forging (metallic forms obtained from dies), machining (for complex geometries), and welding (local heating and fusion to produce complex parts) may additionally modify the physical properties of the metal or alloy being used. The materials currently used in the production of medical devices include stainless steels, cobalt-base alloys, titanium-base alloys, platinum-base alloys, and nickel– titanium alloys. Steels were the first modern metallic alloys to be used in orthopedics and initial problems with corrosion were overcome by modifying the composition of the steel with the addition of carbon, chromium, and molybdenum. Carbon was added at low concentrations (ca. 0.03–0.08%) to initiate carbide formation, while the addition of chromium (17–19%) facilitated the formation of a stable surface oxide layer and the presence of molybdenum (2.0–3.0%) was found to control corrosion. The compositions of stainless steels used can vary widely. Table V shows the limits for the chemical compositions of three different alloys containing eleven different elements together with the mechanical properties for the samples after annealing and cold working. There are at least four compositions of cobalt-base alloys in use which are similarly designated by code numbers such as F75, F90, F562, and F563. Again, these differ in the relative composition of the following elements: manganese, silicon, chromium, nickel, molybdenum, carbon, iron, phosphorus, sulfur, tungsten, titanium, and cobalt. These alloys are used because of their superior

strengths but they are difficult to machine and much more expensive to produce. Titanium-base alloys are found in many commercial medical devices and they are also used as coatings. For dental implants bone is found to grow best in the presence of titanium or materials coated with titanium where surface roughening during manufacture is also found to improve the performance. Platinum-base alloys are used primarily in electrodes for electrical stimulation for although they show excellent strength and corrosion resistance they are very expensive materials to produce and machine. Figure 6 shows the clinical uses of metals in the human body. In many instances metallic implants have to be fixed in to the body and the implants must be compatible with the fixative which may be metallic (screws), ceramic (screws and other components) and/or polymer phases (e.g., glue). In the design of replacement components with high strength it is important that the compatibility of all of the biomedical components is required and investigated before novel implants are placed in the human body. D. Ceramics During the last 40 years a revolution in the use of ceramics has occurred. The revolution is the development of specially designed and fabricated ceramics, termed “bioceramics” when used in the body for the repair and reconstruction of diseased, damaged, and “worn out” parts

TABLE V Chemical Composition and Tensile Strength of Standard Stainless-Steel Alloys Used in Biomedical Applications F55(%) Composition Element Carbon Manganese Phosphorus Sulfur Silicon Chromium Nickel Molybdenum Nitrogen Copper Iron Ultimate tensile strength Annealed Cold-worked

F138(%)

Grade 1

Grade 2

Grade 1

Grade 2

F745(%)

0.08< 2.0< 0.03< 0.03< 0.75< 17–19 12–14 2.0–3.0 0.1< 0.5< Balance

0.03< 2.0< 0.03< 0.03< 0.75< 17–19 12–14 2.0–3.0 0.1< 0.5< Balance

0.08< 2.0 0.025< 0.01< 0.75< 17–19 13–15.5 2.0–3.0 0.1< 0.5< Balance

0.03< 2.0 0.025< 0.01< 0.75< 17–19 13–15.5 2.0–3.0 0.1< 0.5< Balance

0.06< 2.0< 0.045< 0.03< 1.0 17–19 11–14 2.0–3.0

Balance

MPa

MPa

MPa

480–515 655–860

480–515 655–860

480>

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FIGURE 6 Clinical use of metals in the human body.

of the human body. Figure 7 shows many of the clinical applications of ceramics together with the materials used. Most applications relate to the repair of soft and hard tissues but ceramics are also used in the formation of replacement heart valves. Ceramics include inorganic materials such as carbons, silica, and metal oxides in crystalline and glassy phases and salts such as calcium hydroxyapatite, found naturally in bone and enamel. Ceramics are stiff, brittle materials that are generally prepared by high temperature methods and the resulting materials are insoluble in water. The materials have high strength and hardness but they are only able to deform minimally under loading and therefore fracture easily. The materials are made in a variety of physical forms such as powders, coatings, and bulk phases. They can show a range of crystallinity from single crystals (e.g., sapphire), polycrystalline materials (e.g., alumina and hydroxyapatite), glasses (e.g., Bioglass® ), glasses mixed with other ceramics (e.g., A/W glass–ceramic).

They can also be used in polymer ceramic composites (e.g., polyethylene–hydroxyapatite). The specific material form used depends on the application. For example, single crystal sapphire is used as a dental implant because of its high strength. A/W glass–ceramic is used to replace vertebrae because it has high strength and bonds to bone. Bioactive glasses have low strength but bond rapidly to bone and are therefore used in the repair of bony defects. It should be noted in all applications, implants must remain within the human body for many years and the long-term stability of the materials used in the biological aqueous/ saline environment must be considered. 1. Conventional Ceramics These include oxides and salts of metallic elements and silicon together with carbons. The use of oxide phases in implants eliminates the problem of metallic corrosion in a saline environment with high-purity alumina (Al2 O3 )

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FIGURE 7 Clinical use of inorganic bioceramics in the human body.

being one of the first oxide materials to be developed for use in load-bearing orthopedic prostheses. Cermamics based on alumina mixed with beta-silicon nitride (βSi3 N4 ) known as Sialon® are also used with the mixture having higher fracture toughness than either component on its own. Zirconia (ZrO2 ) and yttrium and magnesium stabilized zirconias are also used when applications require a fully densified material. Machinable glass–ceramics containing mica as the main crystal phase such as Macor® with a base glass composition of 47.2 SiO2 , 8.5 B2 O3 , 16.7 Al2 O3 , 14.5 MgO, 9.5 K2 O, and 6.3 F− , and DICOR® , which contains significant amounts of the alkaline earth oxides are used in the production of dental crowns be-

cause the materials show excellent machinability, translucency, and good bending strength. BIOVERIT® II is a phlogopite-type glass–ceramic with two major crystal phases, mica and cordierite, present in the glassy ceramic. The translucency, mechanical properties, and thermal expansion coefficient can all be regulated in accordance with the required medical usuage. The material is biocompatible but not bioactive and is used in middle ear implants and in dental work. BIOVERIT® I has a different composition being a mica-apatite glass–ceramic and offers the possibility of a machinable glass together with bioactivity. Both of BIOVERIT® materials have successfully been used in head and neck surgery and in orthopedic surgery.

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186 A range of carbons with different structure are used in the production of artificial heart valves and orthopedic applications. Glassy carbons (showing different degrees of short-range order) and pyrolytic carbons are both used. Glassy carbon manufactured by pyrolysis of a semicoke retains some porosity and has relatively good wear resistance. Silicon carbide/carbon composites are prepared by impregnating a porous carbon with liquid silicon. Carbon-reinforced carbons and other reinforced carbons are made by impregnating organized filaments with a carbon filler, compressing, heat-treating, and carbonizing or graphitizing. Composites such as CFSiC admixed with SiC lead to materials with Young’s moduli close to that of bone. They exhibit biological and mechanical stability and they are being investigated as new candidates for hip joint replacement rather than the metal or oxide phase conventionally used. The other ceramic widely used are phosphate salts of calcium, with the chosen phase usually being hydroxyapatite. This material is conventionally prepared by thermal methods at temperatures well in excess of 1000◦ C. As a result of their preparation at high temperatures, the salts are carbonate free and are made up of much larger and more perfect crystals than those found in biological apatite minerals including bone. The imperfect crystalline structure of bone mineral leads to the natural material being soluble and reactive with respect to body fluids. In contrast, the synthetic materials are much less reactive than those found in living tissue and problems with biocompatibility can arise. In all cases, solid nonporous implants do not allow for biofilm or cell attachment at any site other than the bulk surface. If porous implants of the previously mentioned materials can be made interfacial stability between the implant and tissue will increase as cells will migrate into the structure. For example, bone will grow in pores greater than 100 µm in diameter and a blood supply can be maintained throughout a material with such porosity. However, such materials show reduced strength and toughness. A compromise is the application of porous ceramic coatings to metals as in-growth of, for example, bone can occur at the porous interface with the mechanical load being carried by the bulk–metal substrate. Problems with such implants usually arise from any incompatibilities between the metal substrate and ceramic film rather than between the ceramic and the natural tissue which overgrows the implant. 2. Bioactive Ceramics Bioactive ceramics are defined as those which are nontoxic and biologically active and that favor the development of an interfacial bond, 0.01 to 200 µm thick between

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implant and tissue. In all cases interfacial dissolution of the ceramic phase in the saline environment of the body leads to modifications in surface chemistry which affect the precipitation of the calcium phosphate phase prior to cell growth and the more general incorporation of the implant. Four major categories of materials have been developed. These include: Dense hydroxy(l)apatite (HA) ceramics, Bioactive glasses, Bioactive glass–ceramics, Bioactive composites. Bioactive glasses are conventionally prepared by the traditional methods of mixing particles of the oxides or carbonates and then melting and homogenizing at temperatures of 1250–1400◦ C. The molten glass is cast into steel or graphite molds to make bulk implants. A final grind and polish are often required. Powdered materials are produced by grinding and sieving the ceramic to achieve the desired particle size characteristics. The chemical components of bioactive glasses include CaO, P2 O5 , Na2 O, and SiO2 . The bonding to bone has been associated with the formation of hydroxyapatite on the surface of the implant. Although a range of compositions can be used (up to 60% silica), an even narrower range of compositions are found to bond to soft tissues. A characteristic of the soft-tissue bonding compositions is the very rapid rate of hydroxyapatite formation. This has previously been attributed to the presence of Na2 O or other alkali cations in the glass composition which increases the solution pH at the implant-tissue interface and thereby enhances the precipitation and crystallisation of hydroxyapatite. The rate of hydroxyapatite formation has also been shown to be strongly dependent on the ratio of SiO2 , the glass network former to Na2 O, the network modifier in the glass. When the glass contains over 60% SiO2 or more, bonding to tissues is no longer observed. The solubility and chemistry (including diffusion of Na+ ions, for example, by the addition of La2 O3 ) of the glass phase can be modified by the incorporation of other phases. Problems which associated with the conventional high temperature method of production arise from: 1. Highly charged impurities such as Al3+ , Zr4+ , Sb3+ , Ti4+ , Ta5+ etc., which can be picked up at any stage of the preparation process. The incorporation of impurity ions leads to dramatic reductions in bioactivity. 2. Processing steps such as grinding, polishing etc. all expose the bioactive powder to potential contaminants. 3. There is a compositional limitation on materials prepared by the conventional high temperature methods due to the extremely high equilibrium liquidus temperature of SiO2 , 1713◦ C, and the

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extremely high viscosity of silicate melts with high silica content. 4. High temperature processing leads to increased processing costs. 5. The relative expense of sol-gel materials relative to traditional precursors for melting gives increased processing costs. An alternative route to the production of bioactive glasses is to use low temperature processing methods including “Sol-Gel Technology.” Materials can be prepared from colloidal powders and simple molecular alkoxide precursors. The “glass” phase originally forms as a gel that can subsequently be dried by supercritical methods or conventional heating to moderate temperatures of

600–700◦ C. The two methods of solvent removal give a much better control over sample purity. Advantages of the technology are the ability to make materials with a much wider range of composition and/or microstructure through manipulation of the processing parameters. Figure 8 shows reaction pathway for the formation of a sol-gel material and application of the technology to the production of materials in a variety of different physical forms from films and coatings through to powders and glasses. All precursors show different reactivity in the initial stages leading to the formation of sol particles (1–500 nm diameter) and gel structure. Using this technology it has been possible produce produce bioactive glasses from CaO–P2 O5 –SiO2 mixtures. Materials produced by the sol-gel route show much

FIGURE 8 The sol-gel process for the preparation of materials.

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188 higher levels of bioactivity for correspondingly lower levels of calcium oxide within the glass due to porosity generated during sample formation by this route which provides additional sites for mineral nucleation and sites for bone cells. 3. Toward Resorbable Implants Biomaterials which are used to repair the body need to last as long as the patient does. At present this is not the case and some people may face several hip replacement operations, for example, each time there being less bone material (or less healthy bone material) for incorporation of devices. The current life expectancy of such replacements is on the order of 10 years at present. This needs to be doubled on tripled in the future. None of the materials described above is able to address the problem of tissue alteration with age and disease. The skeletal system has the capacity to repair itself, this ability diminishing with age and disease state of the material. The ideal solution to the problem is to use biomaterials to augment the body’s own reparative process. Certain of the resorbable implants such as tricalcium phosphate and some bioactive glasses are based on this concept. Problems which exist with the development of resorbable materials are (a) the products of resorption must be compatible with cellular metabolic processes and (b) the rate of resorption must also be matched by the capacities of the body to process and transport the products of this process. In addition, as the material is resorbed and new material formed, the properties of both phases will alter and compatibility must be maintained at all times. This is difficult to achieve. E. Composites Composite materials are used clinically in order to take advantage of the desirable properties of each of the constituent materials while limiting the undesirable or deleterious properties of the individual phases. Composites cover a wide range of compositions and representative materials are listed in Table VI. Most of what is discussed in this section will relate to bioceramic composites. Bioceramic composites are either bioinert, bioactive or biodegradable. Examples of each of these classes of composite and their applications are given in Table VII. The ceramic phase can be the reinforcing material, the matrix material or both. The incorporation of high strength fibers increases the mechanical strength of the composites while maintaining the bioactivity of the material. In the case of glass doped materials, the fracture toughness of the material increases dramatically and renders materials suitable for dental implantation and hip replacement

Biomaterials, Synthetic Synthesis, Fabrication, and Applications TABLE VI Composites Used in Medical Devices Composite Glass, glass–ceramic or HAP with PMMA Tricalcium phosphate/HAP with PE Glass–ceramic, quartz with BIS/GMA Drugs with various polymers/ceramics Carbon or glass fiber with PMMA and other matrices

Medical device applications Bone cement Bone Substitutes Dental restorations Drug delivery All of the above applications but with increased strength and/or stiffness

therapy. The chemical composition of the fibers can be important in establishing continuity between the metallic component and the coating with titanium being an especially good candidate to achieve such an effect. 1. Composites Based on HAP There are many applications for calcium phosphate bioceramics can be seen in Fig. 7. The form of calcium phosphate used in orthopedic clinical applications is usually based on hydroxyapatite and β-tricalcium phosphate. The materials are widely used in composite formulations together with: 1. Ceramics. Mixed calcium phosphates, calcium sulfates, zinc calcium phosphates, aluminium calcium phosphates, metacalcium phosphates, sodium metacalcium phosphate, calcium carbonate, magnesium calcium carbonate and magnesium carbonate. 2. Biological derivatives. Bone derivatives (autografts, allografts and xenografts), collagen, dura, fibrin, amino acids, polyfunctional acids, inductive factors TABLE VII Bioceramic Composites Category Inert

Bioactive

Resorbable

Examples Carbon fiber reinforced carbon Carbon fiber polymeric matrix materials (polysulfone, poly(aryl) ether ketone Carbon fiber reinforced bone cement A-W glass–ceramic Stainless steel fiber reinforced Bioglass® Titanium fiber reinforced bioactive glass Zirconia reinforced A-W glass–ceramic Calcium phosphate particle reinforced polyethylene Calcium phosphate fiber and particle reinforced bone cement Calcium phosphate fiber reinforced polylactic acid

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(bone morphogenic protein), growth factors (bone, epidermal tissue, cartilage, platelet, insulin). 3. Therapeutic agents. Hormones, antibiotics, chemotherapeutic drugs. 4. Synthetic polymers. Polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCA), polyamino acids, polyethylene (PE) and high molecular weight derivatives, polysulfone, polyhydroxybutyrate. 5. Metals. Titanium-, cobalt-, and iron based alloys. These materials can be developed in the form of particulates with a range of porosities, moldable forms, block forms, scaffolds, fibers, and coatings. 2. Bone Graft Materials Bone graft substitutes are available based on alumina chemistry, silica, synthetic and natural calcium salts (phosphate, carbonate, sulfate, and hydroxide) and these materials combined with natural polymers such as collagen, and synthetic polymers such as PMMA, PHEMA, and UHMWPE. Both sintered and nonsintered materials based on calcium phosphate are available with the nonsintered versions showing greater biocompatibility (simply due to better resorption characteristics!). Materials can be produced with a high degree of porosity thus mimicking natural bone and allowing cells to permeate the implanted material over time. These can be produced from natural corals where the biomineralized skeleton of calcium carbonate is replaced by calcium phosphate. Examples include Interpre 200 and 500 with the materials being nearly nonresorbable. The same coral based materials, can be used in their calcium carbonate form without modification with resorption and replacement by fibro-osseus bone tissue. Calcium sulfate is routinely used as a casting material for fractures and is used for dental repairs together with porous hydroxyapatite granules (Hapset). The calcium sulphate is resorbed and can be replaced with the osseous tissue growing around the HA granules and holding them in place. Another biomaterial which makes use of calcium hydroxide together with PMMA coated with PHEMA is the hard tissue replacement polymer HTR. The implant material consists of PMMA beads which are sintered together to give a porous mass which is then coated with PHEMA and calcium hydroxide. The PHEMA coating absorbs a lot of water and a gel is formed at the surface containing calcium ions. This material is very biocompatible. Other alternatives for implants are based on natural bone rather than the synthetic derivatives. For example, ashed bone can be used in combination with Ultra-High Molecular Weight Polyethylene, UHMWPE for coating on a

porous implant for the purpose of biological fixation in joint repair. UHMWPE is the polymer of choice for the matrix material because of its abrasion-resistance, impact resistance, corrosion resistance and zero moisture absorption. The ashed bone provides the bioactive properties to prevent rejection.

V. THE WAY FORWARD, TISSUE ENGINEERING Although many materials, both synthetic and natural, are used in medical devices and treatments it remains the goal of scientists and clinicians to be able to replace diseased or damaged tissues with living substitutes produced in the laboratory. These substitutes would be available in limitless quantities and be able to avoid rejection due to the body’s own immune system. Tissue engineering is a way forward. Tissue engineering encompasses the study of cellular responses to materials implants, manipulation of the healing environment to control the structure of regenerated tissue, the production of cells and tissues for transplantation into the body, and the development of a quantitative understanding of biological energetics. Engineers, chemists, life scientists, and clinicians all have important roles to play in the furtherance of the discipline. Current areas of interest are the design of biocompatible casings for cell transplants, the development of polymer composites for patching wounds, the generation of scaffolds that guide and encourage cells to form tissues, the building of bioreactors for the large-scale production of therapeutic cells, and the establishment of experimental and mathematical models to predict cell behavior. A. Prevention of Unwanted Tissue Interactions Experimentation in this area began as early as 1933 with the use of synthetic nitrocellulose membranes to encompass cells and prevent an immune response. Current applications of the technology extend to the use of laboratory-grown skin in the treatment of burns and ulcers, the treatment of cancer patients via an increase in their marrow cells with culture external to the body and in the detoxification of liver cells from patients with liver failure. Some materials that are being investigated as immunoprotective coatings are, alginate–polylysine coacervates, polyacrylates, polyphosphazenes, materials based on hyaluronic acid, and cellulose as well as hydrogel membranes directly synthesized on cells. The advantage of synthetic membranes as immunoprotective coatings is that they can be tailored for mechanical strength, biocompatibility, permeation characteristics, and

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190 biostability via modifications to the synthesis procedure. They are being explored for the encapsulation of liver and dopamine-secreting cells for the treatment of liver disorders and nervous system disorders as well as the prevention of scar-tissue formation after surgery. B. Directing Tissue Formation Tissue engineering is causing significant advances to be made in “guided tissue formation.” Isolated cells within a larger body of tissue exhibit little ability to organize themselves and form tissues. However, if cells are in fairly close proximity to one another they can grow and exhibit order in the formation of tissue with particular chemical and biological properties. The goal in tissue engineering has been to create an in vitro environment that would enable cells to organize themselves to form functioning tissues. The intention would then be to implant the artifically created cell structure to create new tissue or organs. In order to do this degradable polymer scaffolds have to be synthesized. Approaches to this have used copolymers of lactic acid and glycolic acid. Controlled porosity can be generated by use of salts and other additives that are later washed out prior to seeding the scaffold with cells. An alternative approach to the generation of porosity is to use carbon dioxide to dissolve the polymer phases and then allow them to reform around the gas bubbles on placement of the carbon dioxide dissolved polymer in an atmosphere of air. Living structural supports hold cells close together using adhesive proteins such as fibronectin and vitronectin which bind reversibly to cell surfaces via a specific amino acid sequence-arginine–glycine–aspartic acid. This enables the cells to adhere to and interact with each other and with collagen and other constituents of the extracellular matrix. A modification of a lactic acid–lysine copolymer has been made with the tripeptide attached to the polymer via an amino group on the lysine. This approach to the synthesis of synthetic polymers with the essential components of natural proteins is being widely investigated. It combines the advantages of synthetic polymers with their desired materials characteristics such as strength, controlled degradation and processibility together with the essential features of biological polymers such as cell recognition and the capacity to control cell differentiation. A goal is to modify the specificity of the interaction between the polymer support and cells of choice. Specific advances in this area have been made for substrates used in bone repair which have been altered by binding peptides comprising lysine–arginine–serine–arginine to the surface. This allows specific interaction with osteoblasts rather than endothelial cells and fibroblasts that are also present in an implant area thus encouraging the formation of new bone.

Biomaterials, Synthetic Synthesis, Fabrication, and Applications

Once a cell-seeded scaffold is implanted into host tissue it must allow blood vessels to grow into the new tissue. This can be promoted by the addition of a slow-release angiogenic growth factor which stimulates growth of new blood vessels. Although a polymer scaffold-cell-growth factor complex as described earlier was thought necessary for effective regeneration of tissue, scaffolds alone, cells alone, and diffusible bioactive factors alone may also serve to allow regeneration of tissue under specific circumstances. Tissues which are being prepared or are proposed for preparation via this route include cartilage, skin substitutes, and dental/orthopedic materials. C. Large-Scale Culture of Therapeutic Cells Conventional methods of cell culture have not been successful in the in vitro culture of cells for transplantation. Novel methods of culture are being devised in order to satisfy the demand for cultured cells. Special bioreactors and optimal, precisely controlled culture conditions are necessary to generate large quantities of therapeutic cells such as bone marrow cells for transplant to cancer patients undergoing chemotherapy. A goal for the future is the culture of stem cells from marrow which have been genetically modified to counter disease. D. Future Directions Tissue engineering has had successes in the blocking of unwanted rejection reactions between implants and host tissues, in the synthesis of polymer or polymer–cell composites for tissue repair without scarring, in the development of tissue cell culture for therapeutic cells and in the growth of simple tissues in the laboratory. Future goals include the development of synthetic strategies to materials for implantation which overcome the bodies natural immune response and to generate “universal” donor cells that could be given to all as their immune characteristics would not be recognized by the host as “foreign.” Learning how to stimulate the regeneration of complex multicellular structures in vivo is important and would allow the regeneration of (potentially) all tissues in situ. An important component of these advances will be the identification of specific cell signalling pathways and their spatial and time based involvement in the generation of organs built up from many tissue types. Practically all tissues are capable of being repaired by tissue engineering principles. Engineers, scientists, and clinicians need to use their understanding of synthetic and natural materials and the way in which the human body functions at the cellular level to develop the next generation of biomaterials and cellular transplants for use in the human body. The field is wide open for innovation in the development of a

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new generation of biocompatible materials for use in the human body.

SEE ALSO THE FOLLOWING ARTICLES BIOCONJUGATE CHEMISTRY • BIOINORGANIC CHEMISTRY • BIOMINERALIZATION AND BIOMIMETIC MATERIALS • BIOPOLYMERS • CERAMICS • CERAMICS, CHEMICAL PROCESSING OF • POLYMER PROCESSING • TISSUE ENGINEERING

BIBLIOGRAPHY Burny, F., and Steinbuchel, A. (1998).“Biomaterials and Biodegradable Polymers and Macromolecules: Current Research and Industrial Applications,” Elsevier.

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191 Combe, E. (1999). “Dental Biomaterials,” Kluwer Academic Publishers, New York. Hubbell, J. A. (1995). “Biomaterials in tissue engineering,” Biotechnology 13, 565–576. Hubbell, J. A., and Langer, R. (1995). Tissue Engineering, Chemical and Engineering News 41–54. Park, J. B., and Lake, R. S. (1992). “Biomaterials, an Introduction,” 2nd Ed. Plenum, New York. Perry, C. C. (1998). “Biomaterials,” In “Chemistry of Advanced Materials” (L. V. Interrante and M. J. Hampden-Smith, eds.), pp. 499–562, Wiley VCH, New York. Ratner, B. D. (eds.). (2000). “Biomaterials Science: An Introduction to Materials in Medicine,” 2nd Ed., Academic Press, New York. Silver, F. H., (1994). “Biomaterials, Medical Devices and Tissue Engineering,” Chapman and Hall. Silver, F. H., and Christiansen, D. L. (1999). “Biomaterials Science and Biocompatibility,” Springer-Verlag. Williams, D. F. (1999). “The Williams Dictionary of Biomaterials,” Liverpool University Press.

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Biomineralization and Biomimetic Materials Paul Calvert University of Arizona

I. II. III. IV. V. VI. VII. VIII.

Structural Biological Materials Structural Proteins Structural Polysaccharides Mineralized Tissues Biological and Synthetic Processing The Process of Biomineralization Biomimetic Materials Applications of Biomimetic Materials

GLOSSARY Biomedical materials Materials for medical implants. May be soft, as in vascular grafts, or hard, as in hip replacement. May be wholly synthetic or of biological origin. Tissue-engineered implants are coated with cells previously removed from the patient and cultured on the implant. Biomimetic (or bionspired) materials Synthetic materials formed using principles derived from the growth of biological tissue. Biomineralization The process of forming inorganic material as part of the growth of biological tissue. Freeform fabrication Family of methods to buiild objects from a three-dimensional computer representation driving some deposition system. As opposed to molding or machining.

Structural materials Materials with the principal function of carrying mechanical load, as opposed to sensors, actuators or electrical conductors.

THE MICROSTRUCTURE AND PROPERTIES of biological tissues are of interest because they influence the morphology and behavior of each species. Biological tissues are also of interest in that they can suggest new designs for synthetic materials and new ways of using synthetic materials to achieve an engineering goal. In surgery, we have the more difficult problem of developing synthetic materials that will work in tandem with animal tissues. The following article contrasts synthetic and biological approaches to processing, to material structure and to the use of materials in design, especially as this applies to mineral-reinforced composite structures.

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194 A key observation concerning biological materials is that they are all composites. At any scale above 10 microns there are no uniform structures in biology. This makes the concept of a “biological material” rather uncertain because structures and properties change with position in the body, with the individual plant or animal, and with time. Thus, we can consider “bone” to be a material, but then distinguish the structures of woven, Haversian, and lamellar bone. We then subdivide the bone into dense and cancellous. Finally, the details of the structure will vary with site. Also, in contrast to assembled machines, sharp boundaries between structural materials are rare so it is quite difficult to define where bone stops and mineralized cartilage or mineralized tendon starts. For these reasons, it is also relatively difficult to characterize tissues and compare their properties with those of plastics, ceramics, or metals. This article will summarize biological polymer matrix materials, discuss mineralized tissues, and then discuss biomimetic composites and ceramics.

I. STRUCTURAL BIOLOGICAL MATERIALS In their survey of biological materials, Wainwright et al. (1986) make a division into tensile materials, rigid materials, and pliant materials. Following a more conventional materials division, we will discuss polymers, ceramics and mineralized polymers, and gels; however, such boundaries are even less clear in biology than in the synthetic world. Two processing-induced limitations should be recognized for structural polymers in biology. First, they are all formed from aqueous solution and so are all very sensitive to plasticization by moisture. In most cases, the properties of the dry material have little relevance because they will only occur in a dead organism. The plasticizing effect of water on biological polymers can be regarded as parallel to the softening effect of increased temperature on synthetic amorphous polymers. As temperature will take a hard polymer through the glass transition into a rubbery state, so will increased water content convert amorphous proteins from glass to rubbery. There is little sense in measuring the mechanical properties of biological materials without defining the water content. Second, the growth process allows a variety of routes to the formation of fibers but there are no simple ways to form a dense isotropic plastic. Thus, even essentially isotropic materials will have a fibrous composite microstructure. Weiner et al. (2000) have argued that many biological structures can be viewed as a search for isotropic properties, or at least orthotropic properties (strong in two dimensions), from fibrous materials. This would reflect the unpredictability of stresses encountered by a structure in a dynamic environment.

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II. STRUCTURAL PROTEINS Silks have been reviewed Kaplan et al. (1997). Many insects and arachnids make extensive use of these protein fibers for a range of purposes. Spiders typically produce five different silks for the radial and spiral parts of the web, for a sticky web coating, for wrapping prey, and for the dragline. The properties variations come from differences in the amino acid sequence of the polymer. Silks are stored in a gland as an aqueous solution, at least some of the time in a lyotropic liquid crystalline state. For the strong silks, shear at the spinneret leads to a change in conformation to a very highly oriented and stiff fiber. The combination of strength, stiffness, and toughness shown by spider dragline silks has led to efforts to characterize, clone, and produce a synthetic version of this material . Bulk crystalline polymers, such as nylon or polyethylene, have an amorphous fraction of 30 to 50% that arises from the inability of entangled polymer chains to become completely ordered. This crystallinity can be controlled to some extent by processing and can be reduced by copolymerization to introduce random irregularity into the chain. Fiber structures are less easy to resolve but behave similarly. In many silks, the structure seems to be blocky, with irregular sections spaced along the chain to define noncrystallizable sections. We are not yet able to resolve the role of these irregular sections in stabilizing the liquid crystalline state and in defining the final structure and properties of the fiber. In all biological polymers, there is a degree of control over the molecular structure that may be very important for properties and cannot be duplicated synthetically. Collagen is the structural material of skeletal animals and has properties that are quite inferior to those of cellulose, chitin, or silk. The key to its use seems to lie in the versatile processability. Soluble procollagen is formed in the cell and exported into the growing tissue. An enzyme cleaves a bulky end section from the molecule to form tropocollagen that organizes into a triple helical structure. These triple helices self-assemble into collagen fibrils that make up the bulk of tensile structures such as tendon and ligament. As it ages, the collagen becomes cross-linked, which increases the stiffness and strength but reduces the toughness. Baer and co-workers (1991) have discussed the structure and properties of collagen in tendon and ligament. Keratin can be seen as a biological answer to the need for a tough plastic equivalent to nylon. As hair and fur, it is a fiber. As epidermis, it is a film, and as hoof or horn, it is tough solid. The structure contains fibrils, which are built from three-stranded ropes of alpha-helical chains in a coiled-coil arrangement. The fibrils are embedded in a cross-linked matrix of amorphous protein that is heavily

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cross-linked by cystines (sulfur–sulfur links). The high sulfur content is a defining characteristic of members of the keratin family and gives burning hair its characteristic smell. The fibrils confer great toughness by converting to a more extended beta-sheet structure under stress. Feather keratin is a beta-sheet structure presumably for providing higher stiffness. Amphibian skin is also a beta-sheet, again presumably to limit water swelling. The best-studied example of keratin is wool, but there is also recent work on hoof keratin.

III. STRUCTURAL POLYSACCHARIDES Vincent (1980) discusses insect cuticle, which is a composite of chitin fibers embedded in a cross-linked protein matrix. Chitin is a polysaccharide, similar to cellulose but with acetylamino substitution. Metabolically, polysaccharides should be less expensive than protein since nitrogen has a much lower abundance in the biosphere than carbon, hydrogen, and oxygen. It is not clear what improvement in properties or processing led the insects to select chitin in the place of cellulose as their main structural material. The layered structures of cuticle, with various sequences of fiber orientation, do strongly resemble the layered structures of carbon-fiber composite laminates. Given that both insects and military aircraft are lightweight, roughly cylindrical systems, the resemblance cannot be accidental. Gunderson and Schiavone (1995) have discussed how insects adopt unbalanced or asymmetric layups that would not be used in synthetic composites. Some of the patterns also involve thick layers oriented along the major structural axes, with many fine layers forming the rotation from one direction to the next, apparently to delocalize shear stresses that would cause delamination. Many layered biological systems resemble cholesteric liquid crystals in the rotation of orientation from layer to layer. This has raised recent interest in whether they actually are liquid crystals, in that the rotation forms spontaneously as a result of interactions between fibers in successive layers . This would occur in a fluid state, which is subsequently embedded in a hard matrix. The core question is really whether the rotation pattern is directly controlled by some form of oriented extrusion during the deposition process or is controlled through the surface chemistry of fibrils deposited in successive layers. The mechanical properties of chitin are difficult to define because large oriented samples are unavailable. In cellulose, many plants contain bast fibers with very highly aligned polymer, allowing us to calculate the stiffness of the polymer and so analyze the properties of wood and other plant materials. The stiffness of cellulose in such fibers, 40 GPa wet and 100 GPa dry, is comparable to

the stiffest synthetic fibers, such as Kevlar® . The polymer chains are wholly aligned with the fiber axis. Plant cells are hollow tubes with spirally wound fibrils. Successive layers go in opposite senses, making a crisscross pattern. The outer and inner layers may be wound at different angles to the rest. This is very like the winding of fibers on a composite pressure vessel, such as a pressurized gas tank. The wood composite structure is designed to retain internal pressure or longitudinal compression with very high energy absorption on fracture as the windings collapse inwards. Plants again have the problem of how to manufacture the polymer without it enveloping and choking the production site. It is laid down by a “track-laying” system, which seems to carry out the polymerization at the cell surface on a moving organelle and which draws glucose for polymerization through the membrane from inside. In wood, hemicelluloses and lignin act as a matrix bonding the cellulose fibers into a composite structure. It is a puzzle that the stiffest, strongest, and cheapest of the biological polymers is not used at all in animals.

IV. MINERALIZED TISSUES In the synthetic world, some applications involve predominantly tensile loading, the wall of a pressure vessel being one example. Much more commonly, parts will be loaded in compression or bending. While strong fibers in the form of a rope can provide excellent resistance to tension, they are of little use in compression. Large animals with an external or internal skeleton will need stiff materials that are more isotropic in their properties and so can withstand the varying stresses that come from moving around and colliding with other objects. Mineralized composites provide improved compressive properties over purely polymer structures. Compared to stiff polymers, minerals can also be formed at a lower metabolic cost for a given level of stiffness, but do increase the overall weight. A wide range of minerals is found in microbes, plants, and animals but silica, calcium carbonate, and hydroxyapatite are the most important. A. Silica Silica occurs as spicules (short reinforcing rods) in sponges. The spicules are typically 10 µm in diameter and 100 µm long and may be simple rods or complex branched structures. They apparently form by aggregation of silica nanoparticles onto a thread of polysaccharide or protein within a vesicle, a membrane-enclosed space inside the organism. The material is amorphous, highly hydrated, and not fully dense. There is one example of a deep-sea

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196 sponge, Monoraphis, that is attached to the seabed by a large silica rod, 1 m long. The structure of this material is a series of concentric layers where the weaker interlayer regions may enhance the toughness of the structure, like a laminated glass windshield. Siliceous diatoms are single-celled prokaryotes, about 100 µm in diameter, enclosed by a porous silica shell, in the form of two overlapping dishes, like a petri dish. The organism multiplies by dividing into two disc-shaped cells and then forming two new dishes back-to-back. The cells then separate, each with one new half shell and one old one. Recent work on the proteins involved in silica mineralization has started to clarify the details of silica deposition in diatoms and sponges. Plants often contain silica as a reinforcing material, and the structure of bamboo has been much studied in this context. Seawater and soil water contain low levels of dissolved silica as silicic acid, which the cell probably binds and transports as a complex with catechols (dihydroxybenzene) and then converts this to silica nanoparticles. B. Carbonate Calcium carbonate is widespread as a protective shell in marine animals, from single-celled coccoliths through coral, gastropods, and bivalves. It also occurs as a reinforcement in the cuticle of crustaceans such as crabs. The use of calcium carbonate for shells, rather than silica, may reflect the greater control of structure available through calcium-binding proteins and through control of crystal morphology with nucleators and growth inhibitors. Silica does occur as a component of many mollusk teeth, as do various iron oxides. There has been much recent work on the formation, structure, and properties of mollusk shell. Most shells are constructed of calcite and aragonite in various arrangements with small amounts of protein, up to about 5% by weight. Higher protein levels would be expected to increase the toughness of the shell but at the expense of reduced stiffness. It can be assumed that particular shell structures are adapted to the particular lifestyles of the animals and the stresses encountered. For instance, it might be expected that a swimming bivalve, such as a scallop, would have a stiffer, lighter shell structure. One of the most studied shell structures is nacre, or mother-of-pearl, which is a layered structure of aragonite plates, 0.5 µm thick by several microns wide. Between each layer of plates is a thin protein sheet that is responsible for nucleation of the aragonite layers and acts as a crack stopper, which gives wet nacre a very high fracture energy. While the details of shell formation are not understood, it is clear that organic content contains proteins capable of selectively nucleating calcite or aragonite in a specific orientation and proteins that inhibit growth of specific crystal

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faces. These work in concert with the supply of calcium and carbonate from the mantle tissue. The mantle adds new shell at the existing edge by extruding over the lip to extend the outer and inner faces. The vaterite form of calcium carbonate often occurs in laboratory crystallization and is occasionally found in shells. There is increasing evidence for precipitation of amorphous, hydrated calcium carbonate as a metastable precursor for the crystal. In some cases, including lobster cuticle and a sponge, stable amorphous calcium carbonate is found. C. Hydroxyapatite The human body is supersaturated for both calcium carbonate and for hydroxyapatite, Ca10 (OH)2 (PO4 )6 , the mineral of bone and tooth. Since the environmental availability of phosphorus is limited, its use as a reinforcement seems peculiar. Possibly bone acts a phosphorus reservoir. Bone is essentially a polymer-matrix composite reinforced with ribbons of hydroxyapatite. The structure and properties of bone are discussed by Currey et al. (1995). A key current question is the way the lamellar structure gives rise to a combination of high stiffness, high strength, and high toughness. Similar synthetic composites tend to break at relatively low strain and so have low toughness. Dentine has a structure similar to bone but with significant differences in the mechanism of mineralization. Tooth enamel is almost wholly mineral with a fibrous structure that forms under the control of a completely different set of mineralization proteins, including amelogenin.

V. BIOLOGICAL AND SYNTHETIC PROCESSING In practical engineering, where cost is very important, there is little sense in designing a part without a very good idea of how it will actually be made. Materials selections are then made once the manufacturing route and basic design have been decided. The same must be true in biology; many interesting structural features may be primarily a consequence of the growth process and only secondarily a source of improved properties. Small numbers of synthetic parts can be made by subtractive processes such as machining but the process is slow and wasteful. Molding is generally cheaper and faster. Hot liquid material is injected into a hollow tool and allowed to solidify. Extrusion, forging, rolling, and other hot processes can be viewed as variants on molding. The sintering of ceramics is a separate case; compacted powder is heated until surface energy drives the slow shrinkage and densification to a solid. Chemical processing is not common in the synthetic world. Thermosetting resins such as

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epoxies are solidified by chemical reaction and there is some exotic reactive processing of ceramics. Generally, chemistry has the problem that small changes in starting conditions or purity can cause big changes in the reaction kinetics, so reliability is poor. In biology, all processing is essentially chemical. Soluble reagents are fed to a site where they combine to make a solid plus dissolved by-products. In the case of epoxies, mentioned above, there is little volume change between the liquid and solid states. In forming solids from solution, there is also a massive shrinkage to be accommodated. The biological equivalent of molding can be seen in the formation of isolated particles within an envelope of lipid membrane. Examples include the formation of silica in sponge spicules and diatoms and calcite in coccolith skeletons. The wall of the “mold” is now permeable to allow reagents in and soluble products out. This approach is suitable for isolated particles, such as the magnetic particles in magnetotactic bacteria and for the wall of a singlecelled diatom. The particles may also be later assembled into a simple framework, as in the sponges. However, this method does not lend itself to strong, dense, load-bearing structures suitable for large plants and animals. A problem with building a large solid object by chemical precipitation is to avoid surrounding and entombing the cellular machinery for providing the reagents and removing the products. The obvious solution is to build the solid layer-by-layer, such that a layer of cells provides material to add to the surface of the growing solid and retreat ahead of it. The layer approach is apparent in the growth rings of trees. Bone, tooth, and shell form in the same way. In wood and skin, it is the cells themselves that become the structural unit by depositing solid cellulose or keratin in or on the cell. Each new layer starts as a new layer of cells. In bone and tooth, the deposition is external to the cells. In the case of bone growth, the cell layer continuously forms new collagenous matrix material. This then mineralizes and converts to hard bone as new matrix is deposited over it. The control system thus promotes crystal growth in layers several microns from the cell surface while not mineralizing the freshly formed matrix. Many proteins are known to be associated with this process control but it is not yet clear how they work together. One natural consequence of this layerwise growth process is that layers of different material can readily be formed within a single solid. As will be seen below, biology makes extensive use of layered structures to add toughness to strong materials. Recent developments in ceramics have also focused on the use of layered structures to add toughness. New methods of freeform fabrication should ultimately allow the production of complex layered structures resembling those of biological materials. A result of chemical precipitation is that it is relatively difficult to make dense structures. The large change in

197 volume as a soluble polymer assembles into fibers or as a mineral precipitates will lead to highly porous structures unless the deposition rate is extremely slow. Any structure forming in a diffusion field will tend to grow towards the source rather than filling in gaps in a layer. Very slow growth will allow equilibration, which will favor dense structures. One solution to this is to lay down a mesh of strong fibers, fill in the pores with a softer matrix, and then arrange for this to slowly expel water and harden chemically. This kind of process is seen in wood and in cuticle and very similar issues occur in the formation of carbon–carbon composites. For such reasons, biological materials must be composites.

VI. THE PROCESS OF BIOMINERALIZATION It has long been recognized that most biological tissues mineralize by precipitation in an existing organic matrix that controls crystal form, size, and orientation. Originally, it was thought that the key step would be nucleation on a protein where a suitable spacing of charged groups would match the crystal lattice to be formed. This picture was supported by the observation that acidic proteins extracted from mollusk shell would inhibit crystal growth of calcium carbonate from a protein solution but would nucleate crystal growth if the protein were immobilized on a surface. Since then the picture has become much more complicated. Studies on nucleation at Langmuir monolayers and on self-assembled monolayers have shown that surface ionic charge is important but there is no strong evidence for lattice matching. While the nucleation effect can be important in growing surface-attached mineral films, it seems to work only in a window of concentration just below that at which nucleation occurs readily in solution. This window can sometimes be widened by adding growth inhibitor to the solution. The films that form often seem to be limited in thickness, contrary to the reasonable expectation that growth would readily continue once a mineral layer had covered the substrate. In the case of molluscan nacre it has been shown that some species, at least, nucleate each new layer of aragonite by growing through holes in a protein layer, rather than via a nucleating protein as had been thought. Studies of protein synthesis during biological mineralization have shown that large numbers of proteins are being produced during the growth of tooth or bone. This gives us the problem of too much information, as it is not at all clear why so many are needed and what they all do. It is clear that much of the control is via inhibition of growth on specific crystal faces, leading to control of crystal shape and orientation. At first it might seem strange that a protein can bind so specifically to one crystal face, but a difference in binding strength may be all that is needed to change the relative growth rates of two crystal faces

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198 and this will lead to a change in crystal habit. One essence of control is that signals can be turned on and off. Proteolytic enzymes which degrade specific inhibitors can also be expected to be part of a mineralization process. Studies have also demonstrated that amorphous calcium carbonate does occur in some species and is a transient mineral in some others. This structure presumably results from high levels of incorporated protein in the structure. Other work on solution growth has shown that metastable complexes of calcium and acidic polypeptide can be important in growth of carbonate films. It has long been known that silica species in solution will promote precipitation of hydroxyapatite on many substrates. Again, some metastable complex is presumably involved. De Guire et al. (1998) have studied biomimetic growth of various minerals on substrates treated with self-assembled monolayers. They have shown that some cases seem to correspond to growth by addition of individual ions, while others involve colloidal attraction of preformed clusters to the surface and aggregation. Matijevic has shown that apparently crystalline, faceted particles of many minerals formed from dilute solution are actually aggregates of clusters. Thus, many mineralization processes in complex solutions may involve an intermediate cluster or polymeric state. One striking example of the complexity of biomineralization is the fact that bone is largely mineralized by hydroxyapatite entrained in collagen fibrils, where it is believed to nucleate at the acidic terminal regions of collagen triple helices. Many synthetic studies have produced the mineral on collagen fibrils but none has produced mineral in the fibrils. We are missing some key aspect of the process. It should also be kept in mind that mineralization is a process that occurs in space and time. Mineralized tissue is generally formed by a layer of cells that sequentially deposit organic matrix and mineral and move back. There is a structural gradient away from the cell surface; some species will act locally while others must diffuse some way to their site of action. In bone formation, matrix vesicles also provide the cells with the possibility of delivering species into the mineralizing zone, several microns from the cell surface. In mollusk shell, day–night cycles may also provide the structural sequence. Studies of “flat pearl” have also shown the progression of structure formation on a glass surface embedded under the mantle of a mollusk. Most studies of biomimetic mineralization have used constant precipitation conditions.

VII. BIOMIMETIC MATERIALS The concept of biomimesis has long been used in chemistry in the context of compounds with enzyme-like catalytic action. Since the mid-1980s it has been applied to

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materials, particularly with a view to producing ceramic and composite materials with improved toughness, analogous to shell, tooth, and bone. There was some discomfort in the materials community with the idea that we wanted to mimic biological materials, in the sense of producing an indistinguishable copy. However, mimesis more generally refers to copying some essential aspects of a thing rather than duplicating or faking it. The phrase “bioinspired materials” is also used to express the idea in more familiar terms but is grammatically less desirable as it is a Greek/Latin hybrid. The field has now spread to include a group of loosely linked goals in new materials and processes, which are surveyed below. It should be kept in mind that materials development is considerably upstream from the development of new products. It is quite typical for new materials to find their way into commercial products about 20 years after their discovery. Examples include Kevlar® , high-Tc ceramic superconductors, piezoelectric polymers, and gallium arsenide. It is also typical for the first applications to be quite different from those originally proposed and for their impact to be modest compared to that suggested during the initial excitement. A personal view of the status and prospects for biomimetic materials will be given at the end of this article. A. Polymers The core difference between the proteins and synthetic polymers is that protein synthesis provides total control of the sequence of units along a chain while the best-controlled polymerizations can only provide several blocks of functional units on a chain. One obvious goal is the formation of synthetic polymers with enzyme-like catalytic activity. Many enzyme-active sites have an array of active groups held in close proximity so as to interact with the substrate (target molecule) and to reduce the activation energy for reaction by a precise spatial array of ionic or hydrogen-bonding interactions. To achieve such a precise spacing of active groups on a synthetic polymer would require a rigid structure, which would in turn normally render the material insoluble and so inactive. Recent studies of dendrimer molecules with highly branched structures may lead us to the required combination of flexible and soluble outer structures combined with a highly structured core. Many proteins also go through large changes in shape in response to binding of substrates or other energy inputs. This seems to require a structure where two well-defined conformations are closely balanced in energy so the molecule can flip from one to another. Such a change is not likely in a wholly flexible system but again some subtle combination of flexible segments and rigid units is required. We still have much to learn in these aspects of macromolecular design but are acquiring both the synthetic tools and the understanding.

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As revealed by studies of silk, structural proteins include regular and random regions in the chain structure which would be expected to give rise to crystalline and amorphous material. In contrast, the amorphous component of synthetic polymers arises from entanglements of the coiled chains that cannot be resolved during the crystallization process. New polymer properties may be achievable once we can design polymer chains with such controlled sequences. There has been a report of enzyme-like activity in a block copolypeptide, which enhances the rate of hydrolysis of tetraethoxysilane (TEOS, a standard reagent in sol-gel chemistry) as a suspension in water. If this block structure is a sequence of units of a hydrophilic amino followed by units of a hydrophobic amino acid, it would be expected to be active at a water–solvent interface. The morphology of the silica that forms is dependent on the structure of the copolymer. This system is biomimetic both in the sense of employing a polypeptide catalyst and in the sense of it functioning in a multiphase system, since biological processes rarely occur in homogeneous solutions. Many tissues, such as cartilage, arterial wall, and the walls of soft marine organisms, are swollen polymer structures. Swollen polymers, such as plasticized polyvinylchloride, do occur in artificial structures but they are usually avoided because loss of plasticizer leads to shrinkage and cracking. Even the swelling of wood with changes in humidity is a major impediment to its use in structures, though here years of experience have taught us how to design around it. Skin does change in volume and properties as it takes up or loses water. The structure of amphibian skin keratin is apparently different from mammalian keratin for this reason. There have been many suggestions that designers should make more use of soft structures. These could take the form of composites of hard fibers with rubbers, in which case tires and reinforced plastic tubing could be considered as examples. We could also envision more use being made of liquid-swollen soft structures. For purely mechanical systems, this may not make much sense, but in active systems, such as batteries or muscle-like actuators, a liquid component is necessary and should probably be viewed as a soft material rather than simply as a liquid to be contained. B. Surfactants and Self-Assembly Self-assembly is a hallmark of biological systems, including assembly of protein subunits into holoenzymes, of proteins and nucleic acids into virus particles, and of tropocollagen into collagen fibers. There has been increasing interest in synthetic self-assembly. In addition to the assembly of molecules with complementary hydrogen bonding

to form supramolecular clusters, there are many papers on the assembly of charged polymers and particles to form multilayers and on the assembly of particles or particles and films coated with complementary biological recognition molecules, such as the biotin–streptavidin system. Work at the Mobil Corporation showed that mesoporous silica could be formed by hydrolysis of tetraethoxysilane entrained in a highly concentrated water/silane/surfactant system. In this regime, the three-component mixture forms ordered structures with a range of symmetries. In one hexagonal phase, rods of water are surrounded by surfactant and embedded in a hydrophobic silane matrix. Hydrolysis of the silane under suitable conditions, followed by drying and sintering, results in a porous silica with aligned pores of a few nanometers’ diameter. The growth of the lyotropic liquid crystal precursor is very sensitive to the environment. Ozin and co-workers have shown that complex particle morphologies can result from growth of these mesoporous structures in quiescent solutions as diffusion fields and surface forces interact. Several workers have shown how the direction of the rods or plates of silica can be controlled. Polymers can be introduced to form composite structures that are very reminiscent of some biological composites. This does seem to parallel the proposed importance of liquid crystals in the growth of many biological structures. While one would expect that this approach could be extended to many other material combinations, the rules are not understood. Efforts to form similar structures other than oxides, such as titania, or various crystalline materials, have been only partly successful. Possibly, any rapid or localized conversion process also disrupts the liquid crystalline organization. Stupp and co-workers (2000) have produced a range of amphiphiles that assemble into various ribbon and wedge structures, and the authors have explored their catalytic activity. C. Inorganic Particle Formation Coccolithophores, single-celled marine algae, assemble an external skeleton from single crystals of calcite with very complex shapes. The growing crystal is surrounded by a lipid membrane that controls the growth orientation in the crystal, but it is not known exactly how this is achieved. Sponges and diatoms show similar close control of the shape of silica particles on the micron scale. Magnetotatic bacteria form single-domain iron oxide (magnetite) crystals, with a very closely controlled size of a few nanometers, which then aggregate into magnetic chains (Fig. 1). These biological examples all involve growth within a compartment surrounded by a membrane. For sponge spicules there is an organic template on which the mineral grows. In other cases, there may be specific

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FIGURE 1 Magnetic particles from a magnetotactic bacterium, showing chain formation. [Courtesy of Prof. S. Seraphin, University of Arizona.]

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FIGURE 2 Elongated titania particles formed by impregnation of a stretched two-phase polymer with titanium alkoxide, followed by hydrolysis.

nucleation sites on the membrane surface. This suggests synthetic approaches where inorganic particles are grown within a micromold, a predefined space. There have been many efforts to grow particles in liposomes, spherical shells with a lipid bilayer wall. One solution is trapped when the liposome is formed and precipitation occurs when a second reagent, often base, diffuses in through the wall. Generally, the trapped solution must be quite dilute in order to avoid destabilizing the liposome. As a result, the precipitate therefore only occupies a small part of the internal volume. A method is needed to introduce a continuous feed of both reagents through the membrane. There have also been many efforts to grow particles in multiphase polymer systems, such as block copolymers. For instance, a two-phase polymer can be soaked in one reagent, which selectively absorbs into one phase. A cadmium salt could be taken up by a polyether phase. Subsequent treatment with hydrogen sulfide results in precipitation of cadmium sulfide within the polyether. While the volume fraction of sulfide formed is quite small, repeated cycles can give rise to higher fractions of the mineral. Work on precipitation in lyotropic liquid crystalline amphiphile solutions has led to composites structures. One characteristic of biological minerals is their elaborate shapes. For mechanical reinforcement of soft matrices, a filler should be a high-aspect ratio rod or plate particles. These may grow as a natural outcome of differing crystal growth rates along different crystal axes. How-

ever, most simple minerals are not sufficiently anisotropic to form such elongated particles. The shape of biological crystals is probably controlled by selective inhibition of growth on specific crystal faces. Very elongated silica particles are also formed in lipid vesicles but we do not know how important these are for controlling shape in crystals. While synthetic methods offer many ways of controlling particle size, we lack good methods for controlling shape. One route is to form long micromolds by phase separation in a two-phase polymer, which is colddrawn to elongate the included phase. The included phase is then swollen with a metal alkoxide, which is hydrolyzed to oxide (Fig. 2). D. Tough-Layered Structures Large organisms operate in an environment that subjects them to fluctuating forces, from the action of wind and water on plants and from the locomotion of animals. These fluctuating forces and collisions will often result in local damage that should not lead to catastrophic failure. As a result, many biological tissues contain structural features that add toughness without severely compromising stiffness or strength. In mollusk shells and teeth, toughness may arise from added polymeric layers or from very fibrous structures, as discussed by Heuer. The addition of polymer could be especially effective if the polymer structure is capable

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of a large extension breaking after yield, such as occurs in folded beta-sheet proteins, where the unfolding of the chains leads to a large energy absorption, as has been discussed by Morse (1999). However, polymer layers will also lead to a significant loss of elastic modulus when compared to a wholly inorganic material. Much effort has gone into increasing the strength of ceramic materials by improving their toughness. There was initially much effort devoted to fibrous materials, particularly silicon nitride, but the need for reliable, inexpensive processing methods was not met. Attention then switched to ceramic composites with fibrous reinforcements that would add toughness. Mostly these proved to be unstable or reactive at the temperatures needed for turbine engines, while still being difficult to process. There have been many studies of ceramic/metal layered structures, where the metal layers take the role of the polymers in mollusk shell. Such materials are of interest both for use in engines and for armor. There is also interest in incorporating structured porosity in materials to improve fracture properties or to modify elastic properties such as Poisson’s ratio or piezoelectric response. In the case of metal/ceramic layered materials, the key problem is to absorb as much energy as possible within the metal layers. Hwu and Derby have shown that the major energy absorption is due to metal drawing across

the gap after the crack has passed; however, the extent of deformation occurring during the drawing is limited by the thinness of the metal layers. As a result, substantial increases in toughness were only seen when the volume fraction of metal was quite large. In contrast, mollusk shells show significant toughness with only a small percent of polymer. It is likely that some combination of embedded metal, porosity, and interfacial debonding can give much enhanced toughness or impact resistance to ceramics, but current methods for modeling fracture do not provide enough guidance for these designs and the biological models are only just being interpreted. Studies on the influence of polymers on the crystallization of calcium carbonates in vitro have shown that metastable liquid complexes with anionic polymers can be formed that subsequently transform to calcite. Studies of carbonate biomineralization have shown that amorphous calcium carbonate forms either transiently or as a stable phase. It has also been shown that absorbed proteins modify the fracture properties of carbonates. In particular, fracture surfaces show smooth conchoidal fracture, like glass, rather than the faceted fracture characteristic of normal crystals. This suggests that we have much to learn about the modification of the properties of crystals and amorphous solids by entrained polymer (see Fig. 3). There are parallels with the Lanxide process for

FIGURE 3 Porous interior of a sea urchin spine; calcite single crystal. Polymer coating may serve to prevent fracture from surface damage; entrained polymer may enhance the toughness.

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toughening alumina ceramic by entrained aluminum metal particles. E. Biomimetic Processing Methods Freeform fabrication methods allow objects to be built as a series of layers directly from a three-dimensional computer representation. These allow only one material to be used at a time, or a material plus a soluble support structure. It is clearly feasible to combine several different materials into a single object, which would allow the building of something much closer to an organism. A report on simple robots, which were allowed to evolve in a virtual environment and then were built by freeform fabrication, shows how evolutionary methods might be applied to manufactured objects. To build a crude organism would require resolution at the scale of about 10 µm. Most current freeforming methods allow resolution down to about 100 µm and a limited materials set. Microcontact printing and related methods allow much higher resolution, down to about 1 µm, but effectively are restricted to one layer. There is much current interest in ink-jet printing methods, which could provide the required 10-µm resolution while allowing many layers to be deposited. In both synthetic and biological structures, it is useful to keep in mind a distinction between design and patterning. Phase separation, crystallization, and aggregation processes can give rise to patterns in two and three dimensions on a scale from millimeters to nanometers which reflect the kinetics of the separation and diffusion processes. To form working devices or organisms, we need to build to a nonrecurring design, which may include patterned elements. If we seek to adopt biomimetic processes, we will need to exploit self-assembling structures and patterns but within an overall design. In current silicon technology, photolithographic methods can form two-dimensional designs down to less than 1 µm. Much finer resolutions are achievable in laboratory methods. Three-dimensional designs can be formed to below 1 µm using two-photon methods but commercial freeforming methods are limited to about 100 µm. In biology, there are many examples of patterns forming at the nanometer level but most designs are on the scale of individual cells, a few tens of microns. There are structures, such as sensing hairs, which are much finer but the spacing between them is still on the 10-µm scale. F. Cell Adhesion and Tissue Engineering Biomedical engineering is becoming more concerned with the problems of the long-term biocompatibility of synthetic implants. Material wear and degradation and tissue loss or chronic inflammation due to changing mechani-

203 cal loads will limit the lifetime of most implants to about 10 years. In addition, biocompatibility is not a material property but is very dependent on the specific environment of the implant, in terms of implant site in the body, animal species, and animal age. Strategies to eliminate these problems include the use of biodegradable materials, which will eventually be replaced by natural tissue, and tissue engineered implants, where as much of the device as possible is formed from tissue grown in vitro on a synthetic support before implantation. Tissue engineering imposes a need to understand the interactions between neighboring cells, between cells, and between cells and synthetic surfaces. Cell binding to a biological surface proceeds through a series of stages including physical adsorption, interaction between surface macromolecules and specific binding sites on the surface, reorganization of the macromolecules in the cell membrane to bring more binding sites into contact with the surface, and then specific changes in the cell induced by the surface. At the physical adsorption level, studies of cell attachment to self-assembled monolayers on silicon or glass have shown that attached polyethyleneoxide chains form a structureless hydrophilic barrier layer and will prevent adsorption. Different polar end groups will allow cells to absorb, and printed surface patterns with binding and nonbinding areas can be used to control cell shape. Cells recognize and bind to simple short sequences of amino acids in a protein exposed on a surface. RGD (arginine–glycine–aspartic acid) and RADS (arginine– alanine–aspartic acid–serine) are sequences that can be used to induce strong cell attachment. Thus, suitable polymers can be produced which will promote formation of particular cell types to the surfaces for tissue engineering. In the case of bone implants, there has been much interest in the use of bone morphogenic proteins, now produced from cloned bacteria. One group of a family of cell-signaling proteins are known to induce the development of bone or other tissues when locally released in the body. A similar set of signals induces growth of small blood vessels and is an important factor in the development of many cancers. By allowing implants to release such signaling proteins, it should be possible to speed the integration of the implant; however, much remains to be learnted about the appropriate concentrations, gradients, and timing of these signals. All of these efforts take us in the direction of making synthetic organs that look more like a natural organ transplanted from an identical twin. Belcher and co-workers have recently demonstrated that a phage library, displaying 109 different peptide sequences at the surface, can be used to identify short peptide sequences that selectively bind to inorganic semiconductor surfaces. Sarikaya and co-workers have developed

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204 a similar method for control of gold precipitation using Escherichia coli genetics.

VIII. APPLICATIONS OF BIOMIMETIC MATERIALS Vogel (1998) has discussed the importance of biomimesis in engineering, asking whether it is possible to unequivocally attribute any engineering advance to a biological inspiration. There are no unambiguous examples, and there are cases, such as the attempts to imitate flapping flight, where a biological analogy may have inhibited progress. At the same time, many engineering advances clearly drew some inspiration from biology. In chemistry and materials, studies of the structure and properties of biological materials do suggest alternative approaches to particular problems and illustrate new properties that should be achievable in synthetic materials. Biomimesis clearly will have an important role in new biomedical devices and in new devices that try to combine biological structures or organisms with electronics in sensors and actuators. In structural materials, the obvious place for advance is the introduction of more toughness into synthetic composites and ceramics. In electronics there are not yet many signs of a conjunction between the hard, high-resolution, two-dimensional world of silicon and the soft, larger scale, three-dimensional design of the brain and nervous system.

SEE ALSO THE FOLLOWING ARTICLES BIOMATERIALS, SYNTHESIS, FABRICATION, AND APPLICATIONS • BIOPOLYMERS • GLYCOCONJUGATES AND CARBOHYDRATES • MATERIALS CHEMISTRY • SEPARATION AND PURIFICATION OF BIOCHEMICALS • TISSUE ENGINEERING

BIBLIOGRAPHY Baer, E., Cassidy, J. J., and Hiltner, A. (1991). “Hierarchical structure of collagen composite systems: lessons from biology,” Pure Appl. Chem. 63, 961–973. Baskaran, S., Nunn, S. D., Popovic, D., and Halloran, J. W. (1993). “Fibrous monolithic ceramics,” J. Amer. Ceram. Soc. 76, 2209–2224. Beniash, E., Addadi, L., and Weiner, S. (1999). “Cellular control over spicule formation in sea urchin embryos: a structural approach,” J. Struct. Biol. 125, 50–62. Bertrand, P., Jonas, A., Laschewsky, A., and Legras, R. (2000). “Ultrathin polymer coatings by complexation of polyelectrolytes at interfaces: suitable materials, structure and properties,” Macromol. Rapid Commun. 21, 319–348. Brown, S., Sarikaya, M., and Johnson, E. (2000). “A genetic analysis of crystal growth,” J. Molec. Biol. 299, 725–735.

Biomineralization and Biomimetic Materials Burdon, J., Oner, M., and Calvert, P. (1996). “Growth of oxalate crystals on films of acrylate polymers,” Mater. Sci. Eng. C 4. Calvert, P. (1996). “Biomimetic processing.” In “Materials Science & Technology,” vol. 17B, (R. J. Brook, ed.), pp. 51–82, VCH Publishers; Weinheim. Calvert, P., and Crockett, R. (1997). “Chemical solid free-form fabrication: making shapes without molds,” Chem. Mater. 9, 650–663. Calvert, P., and Rieke., P. (1996). “Biomimetic mineralization in and on polymers,” Chem. Mater. 8, 1715–1727. Calvert, P. D. (1994). “Polymers for New Materials,” Polymer 35, 4484– 4488. Cha, J., Stucky, G., Morse, D., and Deming, T. (2000). “Biomimetic synthesis of ordered silica structures mediated by block copolypeptides,” Nature 403, 289–292. Colgin, M. A., and Lewis, R. V. (1998). “Spider minor ampullate silk proteins contain new repetitive sequences and highly conserved nonsilk-like ‘spacer regions,’” Protein Sci. 7, 667–672. Currey, J. D., Zioupos, P., and Sedman, A. (1995). “MicrostructureProperty relationships in vertebrate bony hard tissues.” In “Biomimetics” (M. Sarikaya and I. A. Aksay, eds.), pp. 117–144, AIP Press, Woodbury, NY. De Guire, M., Niesen, T., Supothina, S., Wolff, J., Bill, J., Sukenik, C., Aldinger, F., Heuer, A., and Ruhle, M. (1998). “Synthesis of oxide and non-oxide inorganic materials at organic surfaces,” Z. Metallk. 89, 758–766. Fan, H., Lu, Y., Stump, A., Reed, S., Baer, T., Schunk, R., Perez-Luna, V., Lopez, G., and Brinker, C. (2000). “Rapid prototyping of patterned functional nanostructures,” Nature 405, 56–60. Frankel, R., Bazylinski, D., and Schuler, D. (1998). “Biomineralization of magnetic iron minerals in bacteria,” Supramolecular Sci. 5, 383– 390. Fritz, M., Belcher, A. M., Radmacher, M., Walters, D. A., Hansma, P. K., Stuckey, G. D., Morse, D. E., and Mann, S. (1994). “Flat pearls from biofabrication of organized composites on inorganic substrates,” Nature 371, 49–51. GiraudGuille, M. (1996). “Twisted liquid crystalline supramolecular arrangements in morphogenesis,” Int. Rev. Cytol. Surv. Cell Biol. 166, 59–101. Gunderson, S. L., and Schiavone, R. C. (1995). “Microstructure of an insect cuticle and applications to advanced composites.” In “Biomimetics” (M. Sarikaya and I. A. Aksay, eds.), pp. 163–198, AIP Press, Woodbury, NY. Hayashi, C. Y., and Lewis, R. V. (2000). “Molecular architecture and evolution of a modular spider silk protein gene,” Science 287, 1477– 1479. Iijima, M., and Moriwaki, Y. (1999). “Effects of ionic inflow and organic matrix on crystal growth of octacalcium phosphate relevant to tooth enamel formation,” J. Crystal Growth 199, 670–676. Kamat, S., Su., X., Ballarini, R., and Heuer, A. H. (2000). “Structural basis for the fracture toughness of the shell of the conch Strombus gigas,” Nature 405, 1036–1040. Kane, R. S., Cohen, R. E., and Silbey, R. (1996). “Synthesis of PbS nanoclusters within block copolymer nanoreactors,” Chem. Mater. 8, 1919–1924. Kaplan, D. L., Mello, C. M., Arcidiacono, S., Fossey, S., Senecal, K., and Muller, W. (1997). “Silk.” In “Protein Based Materials” (K. P. McGrath and D. L. Kaplan, eds.), pp. 103–132, Birkhauser, Boston. Kasapi, M. A., and Gosline, J. M. (1997). “Design complexity and fracture control in the equine hoof wall,” J. Exp. Biol. 200, 1639– 1659. Kroger, N., Deutzmann, R., and Sumper, M. (1999). “Polycationic peptides from diatom biosilica that direct silica nanosphere formation,” Science 286, 1129–1132.

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Biomineralization and Biomimetic Materials Lehn, J. M. (1990). “Perspectives in supramolecular chemistry—from molecular recognition towards molecular information-processing and self-organization,” Angew. Chem. Int. Ed. 29, 1304–1319. Levi, C., Barton, J. L., Guillemet, C., Lebras, E., and Lehuede, P. (1989). “A remarkably strong natural glassy rod: the anchoring spicule of Monoraphis sponge,” J. Mater. Sci. Lett. 8, 337–339. Li, C.-W., and Volcani, B. E. (1984). “Silicification in diatom walls,” Philos. Trans. R. Soc. London B304, 519–528. Lipson, H., and Pollack, J. B. (2000). “Automatic design and manufacture of robotic lifeforms,” Nature 406, 974–978. Lowenstam, H. A., and Weiner, S. (1989). “On Biomineralization,” Oxford University Press, London. Mann, S., Burkett, S., Davis, S., Fowler, C., Mendelson, N., Sims, S., Walsh, D., and Whilton, N. (1997). “Sol-gel synthesis of organized matter,” Chem. Mater. 9, 2300–2310. Miyaji, F., Kim, H., Handa, S., Kokubo, T., and Nakamura, T. (1999). “Bonelike apatite coating on organic polymers: novel nucleation process using sodium silicate solution,” Biomaterials 20, 913–919. Morse, D. (1999). “Silicon biotechnology: harnessing biological silica production to construct new materials,” Trends Biotechnol. 17, 230– 232. National Materials Advisory Board (1994). “Hierarchical Structures in Biology as a Guide for New Materials Technology,” National Academy Press, Washington, D.C. Privman, V., Goia, D., Park, J., and Matijevic, E. (1999). “Mechanism of formation of monodispersed colloids by aggregation of nanosize precursors,” J. Colloid Interface Sci. 213, 36–45. Rajam, S., Heywood, B. R., Walker, J. B. A., Mann, S., Davey, R. J., and Birchall, J. D. (1991). “Oriented crystallization of CaCO3 under compressed monolayers. Part 1. Morphological studies of mature crystals,” J. Chem. Soc., Faraday Trans. 87, 727–734. Raz, S., Weiner, S., and Addadi, L. (2000). “Formation of highmagnesian calcites via an amorphous precursor phase: possible biological implications,” Adv. Mater. 12, 38–40.

205 Schaffer, T. E., Ionescu-Zanetti, C., Proksch, R., Fritz, M., Walters, D. A., Almqvist, N., Zaremba, C. M., Belcher, A. M., Smith, B. L., Stucky, G. D., Morse, D. E., and Hansma, P. K. (1997). “Does abalone nacre form by heteroepitaxial nucleation or by growth through mineral bridges?” Chem. Mater. 9, 1731–1740. Sellinger, A., Weiss, P., Nguyen, A., Lu, Y., Assink, R., Gong, W., and Brinker, C. (1998). “Continuous self-assembly of organic-inorganic nanocomposite coatings that mimic nacre,” Nature 394, 256–260. Sigmund, O., Torquato, S., and Aksay, I. A. (1998). “On the design of 1–3 piezocomposites using topology optimization,” J. Mater. Res. 13, 1038–1048. Simpson, T., and Volcani, B. (1981). “Silicon and Siliceous Structures in Biological Systems,” Springer–Verlag, Berlin. Stupp, S., Pralle, M., Tew, G., Li, L., Sayar, M., and Zubarev, E. (2000). “Self-assembly of organic nano-objects into functional materials,” Mater. Res. Soc. Bull. 25(4), 42–48. Vincent, J. F. V. (1980). “Insect cuticle: a paradigm for natural composites.” In “The Mechanical Properties of Biological Materials” (J. F. V. Vincent and J. D. Currey, eds.), pp. 183–210, Cambridge University Press, Cambridge, U.K. Vogel, S. (1998). “Cats’ Paws and Catapults,” W. W. Norton, New York. Wainwright, S. A., Biggs, W. D., Currey, J. D., and Gosline, J. M. (1986). “Mechanical Design in Organisms,” Princeton University Press, Princeton, NJ. Weiner, S., Addadi, L., and Wagner, H. D. (2000). “Materials design in biology,” Mater. Sci. Eng. C 11, 1–8. Whaley, S. R., English, D. S., Hu, E. L., Barbara, P. F., and Belcher, A. M. (2000). “Selection of peptides with semiconductor binding specificity for directed nanocrystal assembly,” Nature 405, 665–668. Xia, Y., and Whitesides, G. (1998). “Soft lithography,” Ann. Rev. Mater. Sci. 28, 153–184. Yang, S., Yang, H., Coombs, N., Sokolov, I., Kresge, C., and Ozin, G. (1999). “Morphokinetics: growth of mesoporous silica curved shapes,” Adv. Mater. 11, 52–55.

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Bioreactors Yusuf Chisti

Murray Moo-Young

University of Almer´ıa

University of Waterloo

I. II. III. IV.

Introduction Bioreactor Systems Considerations for Bioreactor Design Concluding Remarks

GLOSSARY Austenitic stainless steels Stainless steels with an specific type of nonmagnetic crystal structure. Downcomer The region of a an airlift bioreactor where the gas–liquid dispersion flows downwards. Enzymes Protein molecules that catalyze the various biochemical reactions. Heterotrophic growth Growth in which the carbon and energy for building the biomass are derived exclusively from organic chemicals. Mass transfer Molecular-level transport of any substance through any medium. Organelles Well-defined structures associated with specific functions in a cell. Photomixotrophic culture A culture that obtains a part of the carbon needed for making the biomass from an organic chemical and another part from carbon dioxide via photosynthesis Phototrophic growth Growth in which the carbon needed to make the biomass comes from fixation of carbon dioxide via photosynthesis. Pressure drop Loss of pressure with distance downstream from any point in tubes, channels, and other flow devices. Product (or substrate) inhibition A situation in which

the increasing concentration of the product (or substrate) of a reaction slows down the rate of the reaction by interfering with the enzyme(s) that catalyze the reaction. Protoplast A cell with its wall removed. Reduced substrate A substrate that contains relatively little oxygen within its molecules. Riser The region of an airlift bioreactor where the gasliquid dispersion flows upwards. Solidity ratio The ratio of the swept area to the total projected area of the impeller blades, as viewed from directly overhead an installed impeller. Substrate Any compound that is modified by a cell or an enzyme. Water-for-injection (WFI) Highly purified water that conforms to the WFI specifications of the United States Pharmacopoeia.

A BIOREACTOR is any device or vessel that is used to carry out one or more biochemical reactions to convert any starting material (or raw material or substrate) into some product. The conversion occurs through the action of a biocatalyst—enzymes, microorganisms, cells of animals and plants, or subcellular structures such as chloroplasts and mitochondria. The starting substrate may be a simple

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organic chemical (e.g., sugar and penicillin), an inorganic chemical such as carbon dioxide, or a poorly defined complex material such as meat and animal manure. The product of the conversion may be cells (or biomass), viruses, and chemicals of various kinds. The processes occurring in a bioreactor may be summarized as follows: biocatalyst(s)

are carried out with a moistened solid substrate in the absence of free water, e.g., during composting, making of hard cheeses, and fermentation of cocoa beans for chocolate. Submerged processing is widely used in treatment of wastewater and production of vaccines, antibiotics, and many other useful products. Bioreactors for submerged and solid-state processes are discussed next.

Substrates −−−−−−−−→ products. Many different kinds of bioreactors are available and sometimes a given type may be operated in different ways to obtain different results. The different bioreactor designs are needed to accommodate the great diversity of substrates, products, and biocatalysts and the different requirements of the different bioconversion processes.

I. INTRODUCTION Bioreactors are used in all kinds of bioprocesses, including those for making foods such as soy sauce; those for treating domestic and industrial wastewater; and ones for making vaccines, antibiotics, and many other useful chemicals. Bioreactors that produce microbial cells and cells of animals and plants, are known as fermenters. In addition to cells, a fermenter may also produce other chemicals, or convert (or biotransform) a chemical added to the fermenter, to a different molecule. A fermenter may contain either a single cell type (i.e., monoseptic operation), or a mixed population of different kinds of cells. Fermenters that operate monseptically are designed as sealed units, with barriers that prevent ingress of contaminating microorganisms from the environment. Other types of bioreactors may contain only nonviable entities (i.e., ones that cannot multiply) including cells, isolated enzymes, and organelles obtained from cells. The cells and organelles may be freely suspended in an aqueous medium, or they may be confined by various methods of immobilization. Unlike cells and organelles, enzymes are usually soluble in aqueous media; for repeated use, a soluble enzyme may be retained in the bioreactor by ultrafiltration membranes, or the enzyme may be immobilized in an insoluble matrix.

A. Submerged Culture 1. Mechanically Stirred Tank Bioreactors Stirred tank bioreactors consist of a cylindrical vessel with a motor driven central shaft that supports one or more agitators (Fig. 1). Different kinds of agitators are used in different applications. Microbial culture vessels are generally provided with four baffles placed equidistant around the periphery of the tank. The baffles project into the vessel from near the walls (Fig. 1). The baffles run the entire working height of the vessel and they prevent swirling and vortexing of the fluid. The baffle width is 1/10 or 1/12 of the tank diameter. A gap of about 1.5% of tank diameter is left between the wall and the baffle to prevent stagnation of fluid near the wall. The working aspect ratio of the vessel is between 3 and 5, except in animal cell culture applications where aspect ratios do not normally exceed 2. Often, the animal cell culture vessels are unbaffled. The number of impellers used depends on the aspect ratio of the vessel. The lowermost impeller is located about one third of the tank diameter above the bottom of the tank. Additional impellers are spaced with

II. BIOREACTOR SYSTEMS In most bioprocessing situations cells and biocatalysts are submerged and suspended in a broth that sustains live cultures and dissolves the chemicals that are being modified by the action of the biocatalyst. Bioreactors for submerged processing are generally quite different from those used in solid-state cultivation. Solid-state fermentations

FIGURE 1 Mechanically stirred bioreactor.

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FIGURE 2 Agitators or impellers used in stirred bioreactors: (a) Rushton disc turbine; (b) concave bladed turbine; (c) Prochem Maxflo T hydrofoil; (d) Lightnin’ A315 hydrofoil; (e) Chemineer hydrofoil; and (f) marine propeller.

1.2-impeller-diameter distance in between. The impeller diameter is about 1/3 of the vessel diameter for gas dispersion impellers such as Rushton disc turbines and concave bladed impellers (Fig. 2a, b). Larger hydrofoil impellers (Fig. 2c–e) with diameters of 0.5 to 0.6 times the tank diameter are especially effective bulk mixers and are used increasingly. High solidity ratio hydrofoils (Fig. 2c, d) are good for highly viscous mycelial broths. Animal cell culture vessels typically employ a single large-diameter, lowshear impeller such as a marine propeller (Fig. 2f ). Oxygen is provided typically by sparging the broth with sterile air. In microbial fermenters, gas is sparged below the lowermost impeller using a perforated pipe ring sparger

with a ring diameter that is slightly smaller than that of the impeller (Fig. 1). A single hole sparger discharging the gas below the impeller at the tank centerline is used sometimes. Aeration velocity is usually kept at less than 0.05 ms−1 , or the mixing effectiveness of the impeller will be reduced. In bioreactors for animal cell culture, the aeration velocities are lower, usually less than 0.01 m s−1 , and the gas is sparged such that it does not rise through the region swept by the impeller. Mixing, oxygen transfer, and heat transfer improve with increasing agitation and aeration rates. In low viscosity media, the type of impeller has little effect on the gas–liquid mass transfer rate, so long as the power input per unit liquid volume is kept unchanged.

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FIGURE 3 Bubble columns: (a) the basic design, (b) a column with transverse perforated baffle plates, (c) a column with vertical baffles, and (d) a column with corrugated sheet static mixers for gas dispersion.

2. Bubble Columns A bubble column consists of a gas sparged pool of liquid or slurry (Fig. 3a). Usually, the column is cylindrical and the aspect ratio is between 4 and 6. This basic design may be modified by placing various kinds of internals—e.g., horizontal perforated plates, vertical baffles, and corrugated sheet packings—inside the vessel (Fig. 3b–d). Gas is sparged at the base of the column through perforated pipes, perforated plates, or sintered glass or metal microporous spargers. Oxygen transfer, mixing, and other performance factors are influenced mainly by the gas flow rate and the properties of the fluid. The column diameter does not affect its behavior so long as the diameter exceeds 0.1 m. One exception is the mixing performance. For a given gas flow rate, the mixing improves with increasing vessel diameter. Mass and heat transfer performance improves as gas flow rate is increased. In bubble columns the maximum aeration velocity usually remains less than 0.1 ms−1 . The liquid flow rate does not influence the gas– liquid mass transfer coefficient so long as the superficial liquid velocity remains below 0.1 ms−1 . Bubble columns with recirculation and airlift bioreactors are especially suited to hairy root culture of plant cells. The rootlets tend to grow as an entangled static mass with a doubling time of about 2 days. The fluid flowing past the roots supplies oxygen and other nutrients. A bubble column bioreactor with the hairy root mass confined between two perforated retention plates, is shown in Fig. 4. The nutrient medium flowing into the column may be oxygenated

in a separate column, or air may be bubbled in the column that contains the root mass. External oxygenation is suitable when the conditions (oxygen consumption, fluid residence time) in the column with the root mass are such that the spent medium leaving the column is not totally depleted of oxygen.

FIGURE 4 Bubble column bioreactor for hairy root cultivation.

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FIGURE 5 Airlift bioreactors: (a) draft-tube internal-loop configuration, (b) a split-cylinder device, and (c) an externalloop system.

3. Airlift Bioreactors In airlift bioreactors the fluid volume of the vessel is divided into two interconnected zones by means of a baffle or draft-tube (Fig. 5). Only one of these zones is sparged with air or other gas. The sparged zone is known as the riser; the zone that receives no gas is the downcomer (Fig. 5a–c). The bulk density of the gas-liquid dispersion in the gassparged riser tends to be less than the bulk density in the downcomer; consequently, the dispersion flows up in the riser zone and downflow occurs in the downcomer. Sometimes the riser and the downcomer are two separate vertical pipes that are interconnected at the top and the bottom to form an external circulation loop (Fig. 5c). Externalloop airlift reactors are less common in commercial processes compared to the internal-loop designs (Fig. 5a, b). The internal-loop configuration may be either a concentric draft-tube device or an split-cylinder (Fig. 5a, b). Airlift reactors have been successfully employed in nearly every kind of bioprocess—bacterial and yeast culture, fermentations of mycelial fungi, animal and plant cell culture, immobilized enzyme and cell biocatalysis, culture of microalgae, and wastewater treatment. Airlift bioreactors are highly energy efficient relative to stirred fermenters, yet the productivities of both types are comparable. Heat and mass transfer capabilities of airlift reactors are at least as good as those of other systems, and airlift reactors are more effective in suspending solids than are bubble columns. For optimal gas–liquid mass transfer

performance, the riser-to-downcomer cross-sectional area ratio should be between 1.8 and 4.3 in an airlift reactor. All performance characteristics of airlift bioreactors are linked ultimately to the gas injection rate and the resulting rate of liquid circulation. The liquid circulation velocity depends on the difference in gas holdup (i.e., the volume fraction of gas in the gas-liquid dispersion) between the riser and the downcomer. Liquid velocity is affected also by the geometry of the reactor and the viscosity of the fluid. In general, the rate of liquid circulation increases with the square root of the height of the airlift device. Consequently, the reactors are designed with high aspect ratios of at least 6 or 7, or even in the hundreds. Because circulation is driven by the gas holdup difference between the riser and the downcomer, circulation is enhanced if there is little or no gas in the downcomer. All the gas in the downcomer comes from being dragged in with the liquid as it flows into the downcomer from the riser near the top of the reactor (Fig. 5). Various designs of gas-liquid separators (Fig. 6) are sometimes used in the head zone to reduce or eliminate the gas carry over to the downcomer. Most gas–liquid separators work in one of two ways: either the horizontal flow path between the riser and the downcomer is extended (Fig. 6a) so that the liquid resides for a longer period in the head zone and this provides sufficient time for the gas bubbles to disengage; or the entrance region of the downcomer is expanded in cross section (Fig. 6b) so that the downward flow velocity of the liquid is reduced and it no longer drags gas bubbles into the downcomer. Relative

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cial liquid velocity needed to suspend the solids may be so high that the liquid leaves the reactor much too quickly, i.e., the solid–liquid contact time may be insufficient for the reaction and the liquid may have to be recycled to obtain a sufficiently long cumulative contact time with the biocatalyst. The minimum fluidization velocity—i.e., the superficial liquid velocity needed to just suspend the solids from a settled state—depends on several factors, including the density difference between the phases, the shape and diameter of the particles, and the viscosity of the liquid. 5. Packed Bed Bioreactors

FIGURE 6 Gas–liquid separators for airlift bioreactors: (a) extended length of the flow path in the head zone, (b) enlarged entrance cross section of the downcomer zone.

to a reactor without a gas–liquid separator, installation of a suitably designed separator will always enhance liquid circulation, i.e., the increased driving force for circulation will more than compensate for any additional resistance to flow due to the separator. 4. Fluidized Beds Fluidized bed bioreactors are suited to reactions involving a fluid-suspended particulate biocatalyst such as the immobilized enzyme and cell particles or microbial flocs. An up-flowing stream of liquid is used to suspend or “fluidize” the relatively dense solids (Fig. 7). Geometrically, the reactor is similar to a bubble column except that the cross section is expanded near the top to reduce the superficial velocity of the fluidizing liquid to a value below that needed to keep the solids in suspension (Fig. 7). Consequently, the solids sediment in the expanded zone and drop back into the narrower reactor column below; hence, the solids are retained in the reactor whereas the liquid flows out. A liquid fluidized bed may be sparged with air or some other gas to produce a gas–liquid–solid fluid bed. If the solid particles are too light, they may have to be artificially weighted, for example, by embedding stainless steel balls in an otherwise light solid matrix. A high density of solids improves solid–liquid mass transfer by increasing the relative velocity between the phases. Denser solids are also easier to sediment, but the density should not be too great relative to that of the liquid, or fluidization will be difficult. Liquid fluidized beds tend to be fairly quiescent but introduction of a gas substantially enhances turbulence and agitation. Even with relatively light particles, the superfi-

A bed of solid particles usually with confining walls (Fig. 8) constitutes a packed bed. The biocatalyst is supported on or within the solid matrix that may be porous or a homogeneous non-porous gel. The solids may be ridged, or only slightly compressible. The particles may be randomly shaped (e.g., wood chips and rocks) or they may be uniform spheres, cylinders, cubes, or some other shape. A fluid containing dissolved nutrients and substrates flows through the solid bed to provide the needs of the immobilized biocatalyst. Metabolites and products are released into the fluid and are taken out with the flow. The flow may be upward or downward, but downflow under gravity (i.e., trickle bed operation) is the norm specially if the immobilized biocatalyst requires oxygen (Fig. 8). If the fluid flows up the bed, the maximum flow velocity is limited because the velocity cannot exceed the minimum

FIGURE 7 A fluidized bed bioreactor with recycle of medium.

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lized enzyme reactors and “biofilters” for the treatment of gaseous pollutants. Such reactors are particularly attractive for product inhibited reactions: the product concentration varies from a low value at the inlet of the bed to a high value at the exit; thus, only a part of the biocatalyst is exposed to high inhibitory levels of the product. In contrast, if the catalyst particles were suspended in a well mixed stirred vessel, all the catalyst will experience the same inhibitory product concentration as in the fluid stream that leaves the reactor. 6. Photobioreactors

FIGURE 8 A packed bed bioreactor.

fluidization velocity or the bed will fluidize. The depth of the bed is limited by several factors, including the density and the compressibility of the solids, the need to maintain a certain minimal level of a critical nutrient such as oxygen through the entire depth, and considerations of the maximum acceptable pressure drop. For a given voidage—or solids-free volume fraction of the bed—the gravity driven flow rate through the bed declines if the depth of the bed is increased. Nutrients and substrates are depleted as the fluid moves down the bed. Conversely, concentrations of metabolites and products increase. Thus, the environment of a packed bed is nonhomogeneous, but concentration variations along the depth can be reduced by increasing the flow rate. Gradients of pH may occur if the reaction consumes or produces the H+ ion. Because of poor mixing, pH control by addition of acid and alkali is nearly impossible. Beds with greater voidage permit greater flow velocities through them, but the concentration of the biocatalyst in a given bed volume declines as the voidage is increased. If the packing—i.e., the biocatalyst-supporting solids—is compressible, its weight may compresses the bed unless the packing height is kept low. Flow is difficult through a compressed bed because of a reduced voidage. Packed beds are used especially commonly as immobi-

Photobioreactors are used for photosynthetic culture of cyanobacteria, microalgae, and to a much lesser extent, cells of macroalgae (seaweeds) and plants. Photosynthesis requires light and light stimulates some cultures in ways not seen in purely heterotrophic growth. Because of the need to provide light, photobioreactors must have a high surface-to-volume ratio and this greatly affects the design of bioreactor. The demand for light is reduced in photomixotrophic culture where an organic compound is the major source of carbon for the cells and only a limited amount of photosynthesis (i.e., the fixation of carbon dioxide in presence of light) takes place. Only a few phototrophic microorganisms mainly cyanobacteria and microalgae are cultured on large scale. This kind of mass culture is carried out in photobioreactors open to atmosphere, e.g., in ponds, lagoons, and “raceway” channels (Fig. 9). The latter are widely used and

FIGURE 9 A closed-loop raceway channel for outdoor culture of photosynthetic microorganisms.

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they consist of a closed loop recirculation channel that is about 0.4 m or less in depth. The culture is mixed and circulated by a paddle wheel (Fig. 9) or pumps. The channel is built in concrete and may be lined with plastic. The culture feeding and harvest are continuous, except during the night; however, the channel keeps circulating even during the night. Use of open photobioreactors is limited to only a few microbial species—the astaxanthin producer Dunaliella, Chlorella, and Spirulina. The last two are used mostly as healthfood. These few species can be grown in selective environments (e.g., highly alkaline and saline) that suppress contamination by other microorganisms. A greater variety of algae and tissue cells may be cultured in fully closed photobioreactors; however, like the open raceways and ponds, a closed photobioreactor must have a high surface-to-volume ratio to effectively capture light and this requirement greatly increases the installation and operational expenses of these systems. Closed photobioreactors are used mainly to produce biomass for aquaculture feeds. A closed photobioreactor consists of a light capture unit or photoreceiver, a pumping device to circulate the culture through the photoreceiver, and a gas-exchange column to remove the photosynthetically generated oxygen and provide the carbon source (carbon dioxide). Most useful photoreceivers or photocollectors

are made of ridged or flexible plastic tubing. Glass tubing is used in some cases. The tubes may be arranged into a ladder configuration (Fig. 10a) that may be installed flat on the ground or it may be positioned at a 90◦ angle, as a fence. A continuous run tube may be formed into a serpentine configuration (Fig. 10b) or it may be made into a “biocoil.” The latter is obtained by helically winding a flexible polymer tubing on a cylindrical-shaped frame made of aluminum or other metal bars (Fig. 10c). The culture tubes are 3 cm in diameter. The continuous run length of a single tube depends on the oxygen generation rate and the culture velocity. The length of a single tube is usually 50 m, or less. Several sections of continuous run tubing are installed on a given frame and common headers are used to distribute and collect the broth. The height of the coil may be up to 8 m and the diameter may be 2 m or more. (A larger diameter improves illumination in the region enclosed by the coil. The optimum diameter depends on the height of the coil.) The culture is circulated by a pump or an airlift device. The airlift column or a separate tower is used for gas-exchange. Temperature is controlled by evaporative cooling of water sprayed on the solar receiver. Also, a tubular heat exchanger may be used instead of evaporative cooling.

FIGURE 10 Light capture systems for photobioreactors: (a) tubular ladder, (b) continuous run serpentine, (c) biocoil, and (d) thin channels.

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FIGURE 11 Illumination using optical fibers or waveguides.

Instead of using tubes, the photoreceiver is sometimes made of transparent plastic sheets, as in Fig. 10d. In other cases, a conventional vessel with a low surface-to-volume ratio may be illuminated by using optical fibers to convey light inside from an external source (Fig. 11), but this arrangement is not particularly effective. Irrespective of the design of the photoreceiver and the source of illumination (natural or artificial), light is generally the limiting nutrient in phototrophic culture. Except in optically dilute cultures, exponential growth does not persist for long in photosynthetic microbial culture. Because of light absorption and self-shading by cells in dense culture, light soon become limiting and growth kinetics change from exponential to linear. The depth related decline in light intensity is governed by the Beer–Lambert relationship, as follows: I = exp(−K a XL) , (1) Io where Io is the incident light intensity, I is the intensity at depth L, X is the biomass concentration, K a is the light absorption or extinction coefficient that depends on the pigment content of the cells, and L is the culture depth. Obviously, culture depth (i.e., tube diameter, channel depth) must remain quite shallow, or the local light intensity will become too low to support growth. 7. Other Bioreactor Configurations The basic bioreactor configurations discussed above for heterotrophic growth (i.e., stirred tanks, bubble columns and airlift bioreactors, packed and fluidized beds) are generally satisfactory for a great majority of bioprocessing needs. In addition, some basic configurations have been especially adapted to better suite specific applications. For example, stirred vessels for animal and plant cell cultures employ different designs of impeller compared to ones

used in typical microbial culture. Similarly, some animal cell culture vessels are installed with bundles of microporous polymer tubing for bubble-free oxygen supply to animal cells that are particularly susceptible to damage by bursting bubbles. Oxygen or a gas mixture containing oxygen flows through the tubing and oxygen diffuses into the culture broth through the liquid film held within pores in the tube wall. In other instances, a water-immiscible oxygen carrying liquid, or an oxygen vector (e.g., perfluorocarbons and silicone oils), is used to supply oxygen, as shown in Fig. 12. The carrier fluid is oxygenated in a sparged column by bubbling with air. The bubble-free carrier then circulates through the culture vessel where oxygen is transferred to the broth. The oxygen depleted carrier loaded with carbon dioxide returns to the aeration column (Fig. 12). Other bioreactor designs include the rotating drum fermenter (Fig. 13) with internal baffles. This device is used to culture some suspended plant cells. The drum is filled to less than 40% of its volume and rotated on rollers for mixing. Another bioreactor configuration that is suitable for hairy root cultures of plants is the mist, spray, or fog bioreactor. The static root mass is contained in a chamber that is mostly empty. In this design, the nutrients are supplied as a mist of fine droplets suspended in circulating air currents that penetrate the spaces between the roots. The spray of nutrient solution is produced by using a compressed gas atomizer nozzle or a spinning disc spray device. B. Solid-State Culture Solid-state culture differs markedly from submerged culture. The substrates of solid-state fermentations are particulate solids that contain little or no free water. Steamed rice is a typical substrate. Beds of solids are difficult to agitate and solid-state fermentations do not employ intensive mixing. Small particles with large surface-to-volume ratios are preferred substrates because they present a larger surface for microbial action. However, particles that are too small, and shapes that pack together tightly (e.g., flat flakes, cubes), are undesired because close packing reduces interparticle voids that are essential for aeration. Similarly, too many fines in a batch of larger particles will fill up the voids. For fermentation, the substrate is loosely packed into shallow layers or heaps. Deep beds of substrate require forced aeration with moistened air. Aeration rates may vary widely; a typical range being (0.05– 0.2) × 10−3 m3 kg−1 min−1 . Occasional turning and mixing improve oxygen transfer, and reduce compaction and mycelial binding of substrate particles. Unlike many submerged fermentations, solid-sate processes commonly use mixed cultures. Hygienic processing practices are followed in large industrial operations, but

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FIGURE 12 Bioreactor system for bubble-free aeration through a water-immiscible oxygen vector.

sterility standards that are common in submerged culture production of pharmaceuticals are not attained. Solid-state fermentation devices vary in technical sophistication from the very primitive banana leaf wrappings, bamboo baskets, and substrate heaps to the highly automated koji machines used mainly in Japan. Koji fermentations are widely practiced for making foods such as soy sauce. Koji, or molded grain, is a source of fungal enzymes that digest proteins, carbohydrates, and lipids into nutrients used by other microorganisms in subsequent fermentation. Koji comes in many varieties depending on the mold, the substrate, the method of preparation, and the stage of harvest. Koji for soy sauce is made from soybeans and wheat. Soybeans, defatted soybean flakes, or grits are moistened and cooked in continuous pressure cookers. Cooked

FIGURE 13 Rotating drum bioreactor for submerged culture.

beans are mixed with roasted, cracked wheat. The mixed substrate is inoculated with a pure culture of Aspergillus oryzae (or A. sojae). The fungal spore density at inoculation is about 2.5 × 108 /kg of wet solids. After a 3-day fermentation the substrate mass becomes green–yellow because of sporulation. Koji is now harvested for use in a second submerged fermentation step. Koji production is highly automated and continuous. Processes producing up to 4,150 kg h−1 koji have been described. Similar large-scale operations are used also to produce koji for miso and sake. Some common types of commercial solid-state fermenters are shown in Fig. 14. The static bed fermenter employs a single, static bed of substrate (e.g., steamed rice) located in an insulated chamber (Fig. 14a). The depth of the bed is usually less than 0.5 m. Oxygen is supplied by forced aeration through the substrate. The tunnel fermenter is an adaptation of the static bed device (Fig. 14b). Typically, the bed of solids is quite long, but again no deeper than 0.5 m. Tunnel fermenters may be highly automated with mechanisms for mixing, inoculation, continuous feeding, and harvest of substrate. The agitated tank fermenter uses one or more helical screw agitators mounted in a cylindrical or rectangular tank to agitate the fermenting substrate (Fig. 14c). The screws may move on horizontal rails. The rotary drum fermenter consists of a cylindrical drum that is supported on rollers (Fig. 14d). The rotation (1–5 rpm) of the drum causes a tumbling movement of the solids inside. Tumbling is aided by straight or curved baffles attached to the inside walls (Fig. 14d). Sometimes the drum may be inclined, causing the substrate to move from the higher inlet end to

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FIGURE 15 Rotating disc biofilm reactor. Microorganisms grow attached to the surfaces of the discs that rotate slowly to provide the cells with dissolved nutrients and oxygen.

FIGURE 14 Bioreactors for solid-state fermentations: (a) static bed fermenter, (b) tunnel fermenter, (c) agitated tank fermenter, (d) rotary drum fermenter.

the lower outlet during rotation. Aeration is through the coaxial inlet and exhaust nozzles. C. Bioreactors for Immobilized Enzymes and Cells Immobilized enzyme and cell particles may be used in packed bed bioreactors, or the particles may be suspended in stirred tanks, bubble columns, airlift bioreactors, and fluidized beds, as discussed in an earlier section of this

article. Monolayers of animal cells anchored on small spherical microcarrier particles suspended in stirred bioreactors are widely used in producing viral vaccines (e.g., rabies and polio) and some therapeutic proteins. Similarly, suspended inert particles carrying microbial biofilms are employed in some wastewater treatment processes and other fermentations. Microbial biofilms growing on rotating discs (Fig. 15) are also used to treat wastewater. The discs, mounted on a shaft, slowly rotate through a pool of the water being treated. The dissolved pollutants in the water are taken up by the cells and degraded. The oxygen needed for the degradation diffuses into the biofilm as the disc cycles through the atmosphere. Other bioreactor configurations have been developed specifically for immobilized enzymes and cells. Enzymes immobilized within polymeric membranes are used in hollow fiber (Fig. 16) and spiral membrane bioreactors (Fig. 17). In the hollow fiber device, many fibers are held in a shell-and-tube configuration (Fig. 16) and the reactant solution (or feed) flows inside the hollow fibers. The permeate that has passed through the porous walls of the fibers is collected on the shell side and contains the product of the enzymatic reaction. Also, instead of being immobilized in the fiber wall, enzymes bound to a soluble inert polymer may be held in solution that flows inside the hollow fiber. The soluble product of the reaction then passes through the fiber wall and is collected on the shell side; the enzyme molecule, sometimes linked to a soluble polymer, is too large to pass through the fiber wall. Hollow fiber modules are sometimes used to culture animal cells that are confined to the shell side of the module; the separately oxygenated nutrient solution flows inside the fibers and perfuses the cells on the shell side. In the spiral membrane reactor (Fig. 17), the membrane that contains the immobilized enzyme is rolled into a spiral and confined within a shell. The feed or reactant solution flows in at one end and the product is removed from the opposite end of the cylindrical shell.

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FIGURE 16 Hollow fiber membrane bioreactor.

III. CONSIDERATIONS FOR BIOREACTOR DESIGN A. General Features All bioreactors for monoseptic submerged culture have certain common features, as shown in Fig. 18. The reactor vessel is provided with side ports for pH, temperature, and dissolved oxygen sensors. Retractable sensors that can be replaced during operation are used commonly. Connections for acid and alkali (for pH control), antifoam agents,

FIGURE 17 Spiral membrane bioreactor.

FIGURE 18 A typical submerged culture fermenter: (1) reactor vessel; (2) jacket; (3) insulation; (4) protective shroud; (5) inoculum connection; (6) ports for pH, temperature, and dissolved oxygen sensors; (7) agitator; (8) gas sparger; (9) mechanical seals; (10) reducing gearbox; (11) motor; (12) harvest nozzle; (13) jacket connections; (14) sample valve with steam connection; (15) sight glass; (16) connections for acid, alkali, and antifoam agents; (17) air inlet; (18) removable top; (19) medium feed nozzle; (20) air exhaust nozzle (connects to condenser, not shown); (21) instrumentation ports for foam sensor, pressure gauge, and other devices; (22) centrifugal foam breaker; (23) sight glass with light (not shown) and steam connection; (24) rupture disc nozzle.

and inoculum are located above the broth level in the reactor vessel. The liquid level can be easily seen through a vertical sight glass located on the vessel’s side (Fig. 18). A second sight glass is located on the top of the vessel and an externally mounted light can be used to illuminate the inside of the bioreactor. The sight glass on top can be internally cleaned by a jet of steam condensate. The vessel may be placed on a load cell to obtain a better indication of the amount of material it contains. When mechanical agitation is used, either a top or bottom entering agitator may be employed. The bottom entry design is more common and it permits the use of a shorter agitator shaft, often eliminating the need for support

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bearings inside the vessel. The shaft of the agitator is provided with steam sterilizable single or double mechanical seals. Double seals are preferred, but they require lubrication with cooled clean steam condensate, or other sterile fluid. Alternatively, when torque limitations allow, magnetically coupled agitators may be used thereby eliminating the mechanical seals. An air (or other gas mixture) sparger supplies oxygen (and sometimes carbon dioxide or ammonia for pH control) to the culture. Aeration of fermentation broth generates foam. Typically, 20 to 30% of the fermenter volume must be left empty to accommodate the foam and allow for gas disengagement. Foaming in bioreactors is controlled by a combination of chemical and mechanical methods. Chemical antifoaming agents are commonly mixed with the broth at initiation of fermentation. Further additions of antifoam agent are made from time to time, as needed. Typical antifoams are silicone oils, vegetable oils, and substances based on low molecular weight poly(propylene glycol) or poly(ethylene glycol). Emulsified antifoams are more effective because they disperse better in the fermenter. Excessive use of antifoams may interfere with some downstream separations such as membrane filtrations. Hydrophobic silicone antifoams are particularly troublesome, as they foul membrane filters and chromatography media. The use of antifoam chemicals is minimized by combining it with mechanical breakage of foam. A mechanical “foam breaker” may be installed in the headspace of the fermenter, as shown in Fig. 18. The device in Fig. 18 separates the foam—a dispersion of gas in liquid—into its components by centrifugal action, as explained in Fig. 19. The operation of the foam breaker and the addition of antifoam chemicals are controlled by signals from a foam sensor that extends into the bioreactor from the top. The shaft of the high-speed mechanical foam breaker must also be sealed using double mechanical seals as explained for the agitator. In most instances, the bioreactor is designed for a maximum allowable working pressure of 3.78–4.10 bar (absolute pressure) at a design temperature of 150–180◦ C. The vessel is designed to withstand full vacuum. In North America the design conforms to the American Society of Mechanical Engineers (ASME), Section VIII, Division 1, Boiler and Pressure Vessel Code. Other codes may be acceptable in other locations. The reactor can be sterilized in place using saturated clean steam at a minimum pressure of 2.1 bar (absolute pressure). Overpressure protection is provided by a rupture disc located on top of the bioreactor. The rupture disc is piped to a contained drain. Usually a graphite burst disc is used because it does not crack or develop pinholes without failing completely. Other items located on the head plate of the vessel are nozzles for media or feed addition and for sensors (e.g., the foam electrode),

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FIGURE 19 A mechanical foam breaker. The motor, drive, and shaft assembly are used to rotate the stack of conical discs at a high speed. The foam enters the spaces between the rotating discs at a and is separated into gas and liquid by the centrifugal force. The liquid spins into the bioreactor and liquid-free gas exhausts through the nozzle b. The mechanical seal d prevents leakage into and out of the sterile bioreactor. The seal is lubricated by sterile cooling water c.

and instruments (e.g., the pressure gauge). The vessel is designed to drain completely and a harvest nozzle is located at the lowest point on the reactor vessel (Fig. 18). The reactor is either provided with a manhole, or the top is removable. Flat head plates are commonly used in smaller vessels, but a domed construction of the head is less expensive for larger bioreactors (Fig. 18). The bioreactor vessel should have few internals; the design should take into account the clean-in-place and sterilization-in-place needs. There should be a minimum number of ports, nozzles, connections, and other attachments consistent with the current and anticipated future needs of the process. The bioreactor should be free of crevices and stagnant areas where pockets of liquids and solids may accumulate. Attention to design of such apparently minor items as the gasket grooves is important. Easy to clean channels with rounded edges are preferred. As far as possible, welded joints should be used in preference to sanitary couplings. Steam connections should allow for complete displacement of all air pockets in the vessel and associated pipework, for sterilization. Evan the exterior of a bioprocess plant should be cleanly designed with smooth contours, minimum bare threads, and so forth. The reactor vessel is invariably jacketed. In the absence of especial requirements, the jacket is designed to the same specifications as the vessel. The jacket is covered with chloride-free fiberglass insulation which is fully enclosed in a protective shroud as shown in Fig. 18. The jacket is provided with overpressure protection through a relief valve located on the jacket or its associated piping.

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260 For a great majority of applications, austenitic stainless steels are the preferred material of construction for bioreactors. The bioreactor vessel is usually made in Type 316L stainless steel, while the less expensive Type 304 (or 304L) is used for the jacket, the insulation shroud, and other non-product contacting surfaces. The L grades of stainless steel contain less than 0.03% carbon, which reduces chromium carbide formation during welding and lowers the potential for later intergranular corrosion at the welds. The welds on internal parts should be ground flush with the internal surface and polished. Welds are difficult to notice in high-quality construction. In addition to the materials of construction, the surface finish also requires attention. The finish on surfaces which come in contact with the product material and, to some extent, the finish on external surfaces affects the ability to clean, sanitize, and sterilize the bioreactor and the general processing area. The surface finish has implications on stability and reactivity of the surface, and it may have process implications relating to microbial or animal cell adhesion to surfaces. The mill-finished surface of stainless-steel sheet is unsatisfactory for use in bioreactors. Minimally, the surface should receive a mechanical polish. Mechanical polish is achieved by abrasive action of a sandpaper type material on metal. The surface finish may be specified by grit number, for example, 240-grit polish, which refers to the quantity of particles per square inch of the abrasive pad. The higher the grit number, the smoother the finish. More quantitative measures of surface finish rely on direct measurement of roughness in terms of “arithmetic mean roughness,” Ra, or “root mean square roughness.” Microscopic examination of even a highly smooth mechanically polished surface reveals a typical pattern of grooves and ridges that provide sites for microbial attachment. For example, a 320-grit polished surface will have an Ra of the order of 0.23–0.30 µm. Hence, for internal surfaces of bioreactors, electropolished surface is preferable to mechanical polish alone. Electropolishing is an electrolytic process which preferentially removes the sharp microscopic surface projections arising from mechanical polishing; the result is a much smoother finish. Electropolishing significantly reduces the metal surface area and, hence, the product-metal contact area. The treatment imparts corrosion resistance to stainless steel by removing microscopic regions of high local stress; it creates a passivated steel surface, rich in protective chromium oxide. To attain a suitable electropolished finish, the surface should be previously mechanically polished; however, there is little advantage to starting with a much better than 220-grit (Ra ≈ 0.4–0.5 µm) polished surface. If mechanical polish alone must be used, it should be at least 240 grit, and the direction of polish should be

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controlled to produce a vertical grain for good drainage. The surface should receive a nitric acid wash treatment as a minimum. The orientation of the grain does not seem to be of consequence if the surface is to be electropolished. B. Mixing, Heat, and Mass Transfer 1. Mixing and Shear Effects A minimal intensity of mixing is required in a bioreactor to suspended the biocatalyst and substrate particles, prevent development of pH and temperature gradients in the bulk fluid, and improve heat and mass transfer. Mixing also enhances transfer of nutrients and substrates from the fluid to the biocatalyst particles and helps remove and dilute inhibitory metabolites that may be produced. Mixing is generally provided by mechanical agitation or by bubbling compressed gas into the fluid. Excessively intense mixing is harmful; too much turbulence damages certain cells, disintegrates immobilized biocatalyst pellets, and may dislodge biofilms from carriers. Freely suspended microorganisms are generally tolerant of hydrodynamic forces (or “shear” forces) encountered in bioreactors under typical conditions of operation; however, animal cells, suspended plant cells, certain microalgae, and protozoa are especially prone to shear damage. Forces associated with rupture of sparged gas bubbles are known to destroy animal cells. Damage is minimized by using lower aeration velocities, larger bubbles (diameter ≥ 0.01 m), and supplementation of the culture medium with protective additives such as the surfactant Pluronic F68. Aeration associated power input in bioreactors for animal cell culture is typically kept at less than 50 W m−3 . The power input may be calculated as follows: PG = ρ L gUG , (2) VL where PG is the power input, VL is the volume in the bioreactor, ρ L is the density of the broth, g is the gravitational acceleration, and UG is the superficial gas velocity. The velocity is calculated as the volume flow rate of the gas divided by the cross sectional area of the bioreactor. 2. Oxygen Supply and Carbon Dioxide Removal Animal and plant cells need oxygen to survive. Many microorganisms require oxygen (i.e., they are obligate aerobes) but oxygen may be toxic to others (anaerobes). Some microbes may switch between aerobic and anaerobic growth and are said to be facultative. Sufficiency of oxygen supply is necessary to prevent growth limitation in aerobic cultures. Oxygen is provided usually by sparging the broth with air or some other oxygencontaining mixture of gases. Other specialized methods

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of oxygen supply are used in a few bioprocesses (e.g., Fig. 12). Oxygen is sparingly soluble in aqueous broths and even a short interruption in aeration rate in some microbial fermentations may produce anaerobic conditions that may potentially damage the cells. The precise oxygen requirements of a fermentation process depend on the microorganism, the degree of oxidation of the substrate being used for growth, and the rate of oxidation. Oxygen becomes hard to supply when the demand exceeds 4–5 kg O2 m−3 hr−1 . Many microbial fermentation broths are highly viscous and difficult to mix. This further complicates the transfer of oxygen from the gas phase to the broth. Once the concentration of dissolved oxygen falls below a critical value, the microbial growth becomes limited by oxygen. The critical dissolved oxygen concentration depends on the conditions of culture and the microbial species. Under typical culture conditions, fungi such as Penicillium chrysogenum and Aspergillus oryzae have a critical dissolved oxygen value of about 3.2 × 10−4 kg m−3 . For baker’s yeast and Escherichia coli, the critical dissolved oxygen values are 6.4 × 10−5 and 12.8 × 10−5 kg m−3 , respectively. Animal and plant cell cultures have lower oxygen demands than microbial cells. Oxygen consumption rates for animal cells are in the range of 0.05–0.5 mmol/109 cells per hr. In batch suspension culture of animal cells, the maximum cell concentration typically does not exceed 2 × 106 cells ml−1 . Higher concentrations, >107 cells ml−1 , are attained in perfusion culture without cell retention. Perfused culture with cell retention permits cell densities of around 109 cells ml−1 . In vitro cultured animal cells are generally tolerant of high concentrations of dissolved oxygen, e.g., up to 100% of air saturation; however, the optimal concentration is about 50% of air saturation but may vary with cell type. Oxygen concentrations of >100% of air saturation have been associated with oxidative damage to cells whereas concentrations ≤0.5% of air saturation inhibit the TCA cycle and lead to an enhanced production of lactate. Concentrations <10% of air saturation will limit growth of some cells, but for others oxygen limitation is encountered around 0.5% of air saturation. Plant cells consume oxygen at a rate of (3–15) × 10−5 mol kg−1 DW s−1 and the maximum cell density in suspension culture tends to be 20–30 gDW L−1 . As with dissolved oxygen, concentration of dissolved carbon dioxide influences microbial and cell growth. Too much carbon dioxide is detrimental to most aerobic fermentations but cultures of photosynthesizing microbes may require carbon dioxide in the aeration gas. Also, carbon dioxide is added at roughly 5% (by vol) to gas mixture used in sparging animal cell cultures when the culture broth is buffered with carbonate-bicarbonate system. Nor-

mal mammalian cells need carbon dioxide as a substrate for carboxylation of pyruvate to oxaloacetic acid, but established cell line may not require carbon dioxide. 3. Heat Removal and Temperature Control All fermentations generate heat. In submerged cultures, 3–15 kW m−3 of the heat output typically comes from microbial activity. In addition, mechanical agitation of the broth produces up to 15 kW m−3 . Consequently, a fermenter must be cooled to prevent temperature rise and damage to culture. Temperature is controlled by circulating cooling water in a jacket that surrounds the bioreactor vessel (Fig. 20a). In addition to the jacket, an internal cooling coil (Fig. 20b) becomes necessary in large bioreactors. In some cases, cooling is achieved by recirculating the broth through an external heat exchanger (Fig. 20c). In small vessels, a “ringlet” coil (Fig. 20d) that enters the vessel through a large port on top, may provide sufficient cooling. Heat removal tends to be difficult because, typically, the temperature of the cooling water is only a few degrees lower than that of the fermentation broth. Industrial fermentations are commonly limited by the heat transfer capability. The ability to remove heat depends on the surface area available for heat exchange, the temperature difference between the broth and the cooling water, the properties of the broth and the coolant, and the turbulence in those fluids. The geometry of the fermenter determines the heat exchange area that can be provided. Because metabolic heat generation depends on the oxygen consumption rate, heat removal in large vessels

FIGURE 20 Heat-exchange methods for bioreactors: (a) jacket, (b) full internal coil, (c) recirculation of broth through an external heat exchanger, (d) ringlet coil. Cooling water (CW) enters and exits the heat exchange devices, as shown.

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262 becomes difficult as oxygen consumption rate approaches 5 kg m−3 h−1 . About 0.54 kJ of heat is generated for each millimole of oxygen consumed. Oxygen consumption rate in microbial broths typically ranges from 2 × 10−4 to 1×10−3 kg m−3 s−1 . Typically, microorganisms growing on hexose sugars such as glucose produce about 10.9 MJ of heat per kilogram of biomass formed. For cells growing on highly reduced substrates such as hydrocarbons, heat generation is greater, about 28.5 MJ/kg of biomass produced. These figures are for fermentations in which biomass is the only product. Most microbial and plant cells are cultured at a temperature of between 20 and 30◦ C. Mammalian cells are usually grown at 37◦ C. Insect cells prefer a lower temperature, e.g., 26◦ C. Temperatures of greater than 40◦ C are optimal for certain thermophilic microorganisms. In addition to removing the metabolic heat, a fermenter must provide for heat transfer during sterilization and subsequent cooling. Liquid (or slurried) fermentation medium for a batch fermentation may be sterilized using batch or continuous processes. With batch processes, the medium or some of its components and the fermenter are commonly sterilized together in a single step by heating the dissolved or slurried medium inside the fermenter. For in situ sterilization, steam may be injected directly into the medium or heating may be through the fermenter wall. High temperature (typically 121◦ C) heating during sterilization often leads to undesirable reactions between components of the medium. Such reactions reduce yield by destroying nutrients or by generating growth inhibitory compounds. This thermal damage is prevented or reduced if only certain components of the medium are sterilized in the fermenter and other separately sterilized components are added later. Sugars and nitrogen-containing components are often sterilized separately. Dissolved nutrients that are especially susceptible to thermal degradation may be sterilized by passing through hydrophilic polymer filters that are rated to retain particles down to 0.45 µm. Even finer filters (e.g., 0.2 µm rated particle retention) are available. Heating and cooling of a large fermentation batch takes time that ties up a fermenter unproductively. In addition, the longer a medium remains at high temperature, the greater is the thermal degradation or nutrient loss. Therefore, continuous sterilization of the culture medium into a presterilized fermenter is preferable even for batch fermentations. Continuous sterilization is rapid and it limits nutrient loss; however, the initial capital expense is greater because a separate sterilizer is necessary. A heat exchange system must be able to handle the high heat loads encountered during sterilization by steam and subsequent cooling. Irrespective of the specific heat exchange system used (Fig. 20), the operational details can be quite complex as shown in Fig. 21 for a jacketed

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bioreactor. Valves 1, 10, and 5 in Fig. 21 are control valves that modulate the flow of steam and cooling water; valves 2 and 9 are vacuum breakers that allow the pipework to drain under gravity; valve 3 is a pressure relief valve to protect the jacket and the pipework against pressurization above the safe acceptable limit; and all other valves are pneumatically operated devices that are either fully open or fully closed. For sterilization the jacket is heated by steam. Valves 4–6, 8, and 10 are closed and the pump 7 is off. Valve 11 is opened to drain the jacket. After a short period, valve 11 is closed and valve 12 is opened. Valve 1 is opened to let steam into the jacket. The condensate drains through the steam trap 13. The temperature and pressure of the circuit are monitored at TIC1 and PI1. Cooling is carried out by closing valves 12 and 1; valves 8 and 4 are opened. Cooling water enters the circuit at valve 4, flows up the jacket and out to the cooling water return line (Fig. 21). All operations are generally automated for consistent and error-free control. Animal cell culture may actually require some warming to maintain the temperature at 37◦ C. The circuit shown in Fig. 21 uses a closed recirculation loop to control the temperature. During culture, valves 8 and 6 are opened and the pump 7 is turned on. The water is pumped through a compact plate heat exchanger (PHE) where it is indirectly heated by controlled (control valve 10) flow of steam. The heated water now passes the temperature indicator/controller TIC1 and enters the jacket. The water recirculates via valve 8 and pump 7. Cold water is injected (control valve 5) in the circuit to maintain the temperature. Any excess water leaves the loop via the relief valve 3. In some designs, direct steam injection into the circulating loop may be used instead of the heat exchanger. In small fermenters, the exchanger may be replaced by electric heating. Notice the flexible connections between the fermenter and the temperature control pipework (Fig. 21). These connections are necessary for fermenters that rest on load cells (for weight measurement); the connections allow the fermenter to move freely. Compared to submerged culture, biomass levels in solid state fermentations are lower at 10–30 kg m−3 . Nevertheless, because there is little water and the density of the substrate is relatively small, the heat generation per unit fermenting mass tends to be much greater in solid state fermentations than in submerged culture. Temperature can rise rapidly, again, because there is little water to absorb the heat. Cumulative metabolic heat generation in koji fermentations for a variety of products has been noted at 419–2387 kJ kg−1 solids. Higher values, up to 13,398 kJ kg−1 , have been observed during composting. Peak heat generation rates in koji processes range over 71–159 kJ kg−1 hr−1 , but the average rates are more moderate at

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FIGURE 21 Temperature control circuit on a bioreactor.

25–67 kJ kg−1 hr −1 . Peak metabolic heat production rate during fermentation of readily oxidized substrates such as starch can be much greater than in typical koji processes. In solid-state fermentation the substrate temperature is controlled mostly through evaporative cooling; drier air provides a better cooling effect. Intermittent spray of cool water is sometimes necessary to prevent dehydration of the substrate. Air temperature and humidity are also controlled. Occasionally, the substrate-containing metal trays may be additionally cooled by circulating a coolant, even though most relatively dry and porous substrates are poor conductors of heat. Intermittent agitation of substrate heaps further aids heat removal. Despite much effort, temperature gradients in the substrate do occur, particularly during peak growth. C. Monoseptic Operation, Cleaning, Sterilization Many commercial bioprocesses utilize only pure cultures. Maintenance of monoculture is vital to success of such processes; hence, a bioreactor must be sterilized prior to inoculation and contamination during operation must be

prevented. A contaminating virus or microbe may destroy the culture, reduce productivity, or lead to other unwanted results. Poor design and operation of a bioreactor increase the chance of contamination and cause financial loss. Sterilization with steam at a minimum temperature of 121◦ C is the norm. The sterilization temperature must be attained everywhere in the vessel and continuously held at the requisite value for at least 20 min. After sterilization and during culture the contents of the bioreactor remain fully isolated from the external environment. Air enters the vessel through presterilized hydrophobic membrane filters that prevent ingress of contaminating microbes. Similar filters are located on the air exhaust pipe. These filters are rated for removing particles down to 0.45 µm, or even 0.1 µm. The bioreactor may be sterilized together with the culture medium, or separately. In the latter case, once the bioreactor has cooled, the medium is pumped in through a sterilizing filter that removes any contaminating microbes. Alternatively, the medium is steam sterilized, cooled, and then transferred to the bioreactor through pipework that is fully isolated from the surroundings. After the culture and before next sterilization, the bioreactor must be thoroughly cleaned usually by automated

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264 clean-in-place methods. In-place cleaning does not require dismantling the bioreactor and saves time. Automation assures consistency of cleaning. Cleaning between batches is essential to preventing cross-contamination of products. Also, a clean bioreactor is easier to sterilize. Cleaning is achieved by physical action of high velocity flow, jet sprays, agitation, and chemical action of cleaning agents enhanced by heat. While mechanical forces are necessary to remove gross soil and to ensure adequate penetration of cleaning solutions to all areas, most of the cleaning action is provided by chemicals—surfactants, acids, alkalis, and sanitizers. A generally applicable cleaning scheme for bioreactors utilizes a water pre-rinse to remove gross soil; a hot alkali recirculation step to digest and dissolve away the remaining soil; and a water wash to remove residual alkali. A bioreactor that processes injectable drugs, should be rinsed with a hot water-for-injection (WFI) wash as the final cleaning step. Optional acid wash and sanitization steps may be added in some applications.

Bioreactors

FIGURE 23 Bioreactor operational modes: (a) a continuous flow well-mixed bioreactor, (b) a continuous flow well-mixed bioreactor with biomass recycle.

D. Operational Modes of Bioreactors The way a culture is operated and fed has a profound impact on the outcome of the fermentation. Also, the reactant feeding and reactor operational strategies affect performance of enzyme bioreactors and those that use non-viable biomass as a biocatalyst. Most bioreactors for microbial growth and culture of other cells are operated as batch and fed-batch devices. A batch fermentation is initiated by inoculating a presterilized and cooled medium that is contained typically in a well-mixed bioreactor (Fig. 22a). The medium composition in the fermenter changes continuously as nutrients are consumed to produce biomass and metabolites. The broth is harvested after the designated batch time. The broth volume in a batch fermenter remains essentially constant (Fig. 22a), discounting any evaporative loss. A fed-batch culture is identical to a batch operation, except that a feed is added to the broth continuously or intermittently (Fig. 23b). The volume of the broth increases with time. Also, the feeding rate generally incenses with time, to satisfy the demand of an exponentially increasing cell population. The broth is harvested at the end of the batch period.

FIGURE 22 Bioreactor operational modes: (a) a well-mixed batch bioreactor, (b) a well-mixed fed-batch bioreactor.

In a well-mixed steady-state continuos culture (Fig. 23a), the broth composition does not vary with time and position in the bioreactor. Typically, a continuous fermentation starts as a batch culture and switches to continuous feeding when a sufficient concentration of biomass has been obtained. The feed is added continuously and at a constant rate. The broth volume does not change, as the rate of harvest matches the feeding rate (Fig. 23a). Sometimes, a well-mixed continuos culture may be carried out with recycle of a part of the harvested biomass to the bioreactor (Fig. 23b). This strategy increases the steady state biomass concentration in the reactor, improves conversion of the substrate, and enhances the productivity. Some continuous flow reaction and production schemes use a number of well-mixed reactors in series (Fig. 24a–c). The harvest of one reactor becomes the feed for the next. This arrangement is especially useful when different environmental conditions are needed at different stages of a process, for example, when the requirements for growing the biomass differ from the ones for synthesis of a metabolite by the cells. A multistage continuous array of well-mixed reactors may be fed with different feeds and precursor compounds at different stages, as shown in Fig. 24b. Also, such a series of reactors my employ recycle of the biomass (Fig. 24c) to the first stage, or one or more of the other stages. Continuous flow processes sometimes use a plug flow bioreactor in which there is little mixing of fluid elements in the direction of flow (Fig. 25a). This type of flow is typically achieved in long tubes and channels. The composition of the broth does not change with time at a fixed

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FIGURE 24 Bioreactor operational modes: (a) a series of continuous flow well-mixed bioreactors, (b) use of different feeds at different stages in a series of continuous flow well-mixed bioreactors, (c) a series of continuous flow well-mixed reactors with biomass recycle.

location in a plug flow bioreactor operating at steady state; however, the composition changes with position in the direction of flow. A plug flow bioreactor behaves the same as a series of many continuous flow well-mixed bioreactors (Fig. 24a). In terms of kinetics, a plug flow bioreactor is equivalent to a batch fermenter having the same residence time (i.e., the total time spent by the broth in the bioreactor) as in the plug flow device. A plug flow bioreactor for biomass production must be inoculated continuously, or a portion of the harvested biomass must be recycled to the inlet of the bioreactor (Fig. 25b), to prevent wash out of the culture by the sterile feed. Multipoint feeding is sometimes used in plug flow bioreactors, as shown in Fig. 25c. Continuous flow operation of a bioreactor such that the cells are recycled or retained within the reactor, is sometimes known as perfusion culture. Many different kinds of devices are available for cell retention and recycle. These devices may be located within or outside the bioreactor vessel. External sedimentation tanks (Fig. 23b) are often used to recycle biomass in wastewater treatment processes. External and internal enhanced-rate sedimentors are also employed in animal cell culture bioreactors, as shown in Fig. 26a, b. Because of the small differences in

density and diameter of the viable and nonviable animal cells, the inclined channel sedimentor (Fig. 26b) may preferentially retain viable cells in the bioreactor while allowing many of the nonviable ones to wash out. Another device that is commonly deployed for partial retention of animal cells is the “spinfilter” shown in Fig. 27. A spinfilter

FIGURE 25 Bioreactor operational modes: (a) a continuous plug flow bioreactor, (b) a continuous plug flow bioreactor with biomass recycle, (c) multipoint feeding of a plug flow bioreactor.

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FIGURE 26 Cell retention by sedimentation in perfusion culture: (a) internal sedimentor, (b) inclined channel sedimentor.

is a cylinder formed of wire meshing. The openings of the wire screen are significantly larger (e.g., 25 µm) than the cells which are retained by a hydrodynamic mechanism requiring rapid rotation (e.g., 500 rpm) of the spinfilter. The mostly cell-free spent medium is withdrawn from the zone within the rotating screen. Depending on the specifics of design and operation, a spinfilter may preferentially retain the viable cells. Also, a noninvasive acoustic sedimentation device has been developed and this is installed on the outside of the bioreactor harvest pipe. This device uses sound waves to aggregate the cells which sediment back into the bioreactor vessel.

FIGURE 27 A spinfilter device of cell retention in perfusion culture of animal cells.

Spinfilters and sedimentation devices do not retain all cells within a bioreactor and some washout occurs. Total retention of biomass is feasible by using an external microfilter to continuously harvest a cell-free stream, as shown in Fig. 28. The cells are returned to the bioreactor. The bioreactor operational schemes identified here are the ones most commonly used; other variations on these schemes are encountered occasionally.

E. Medium Composition The composition of the nutrient medium used to grow cells determines the rate of production, the type of products produced, and the yield of biomass and products. The medium also influences the cost of production, the quality of the product, and factors such as the broth rheology. The medium must provide all the elements that compose the product and the biomass. These elements (e.g., C, N, O, S, P) must be provided in a suitable form and ratios that are designed to achieve specific effects. The growing cells may require additional complex organic molecules (micronutrients) that they are unable to synthesize but that are essential to growth. In addition, the medium may contain specific precursor compounds, the substrates that are being biotransformed, and inhibitors or inducers of specific enzymes. While the medium needs many components, care is necessary to prevent contamination with others that may affect the process performance. For example, presence of iron in Aspergillus niger fermentation broth greatly suppresses the production of citric acid. Proper formulation of the medium is also important in cell-free enzyme reactors, where the rate of reaction and the extent of substrate (or/and product) inhibition depend on the composition of the reaction medium. Some enzyme reactions are carried out in organic solvents containing small amounts of water.

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FIGURE 28 Total recycle of biomass by using a microfiltration device.

Medium design for viable culture needs to consider the osmotic pressure of the broth as an important variable. Osmotic pressure of a solution is a measure of the concentration of dissolved molecules and ions present as individual particles. All cells are susceptible to osmotic pressure of the surrounding medium, but cells without walls (e.g., animal cells, protoplasts) are especially susceptible to osmotic stress related damage. When the suspending fluid has a higher osmotic pressure than the cells, water is drawn out of the cells and it may become so dehydrated that metabolism ceases. Incidentally, this is why high salt and high sugar media often have a preservative action, e.g. in pickling of vegetables. When the osmotic pressure is low compared to that in the cell, the cell takes up water and may burst. Osmotic shock produced by rapid dilution is sometimes used to rupture cells, especially animal cells. Osmotic pressure also influences plant cell suspension culture. Media formulations do not generally specify the osmotic pressure; instead, the number of dissolved particles is given as osmolarity or osmolality. Osmolality is the number of moles of particles per kilogram of solution whereas osmolarity is the number of moles per liter of solution. One mole of particles is an osmole, abbreviated as Osm. Animal cell culture media have an osmolality of 280 to 320 mOsm kg−1 to conform to the osmolality of serum (290 mOsm kg−1 ). Osmolality is not easily calculated especially when a medium has many components and the degree of dissociation is not known. In such cases, osmolality is estimated from measurements of freezing point depression and other colligative properties (i.e., those dependent on the concentration of dissolved particles). In cell culture media, sodium chloride is used for adjusting osmolality to the requisite value. F. Kinetics, Productivity, and Bioreactors Design and performance analysis of a bioreactor are inseparably linked with the kinetics of the bioreaction for which the reactor is intended. Some common bioreactions include growth of microorganisms and other cells, and re-

actions involving cell-free enzymes. Essential aspects of kinetics for bioreactor design are discussed here.

1. Cell Growth Once a properly formulated and sterilized medium has been inoculated with a seed culture or inoculum, the cells grow and multiply. In a batch culture, the cell or biomass concentration increases with time as shown in Fig. 29, which is typical. A short lag phase of little or no growth is followed by a period of exponential growth. The lag phase is an adaptation period in which the cells become acclimatized to a new environment. The length of the relatively unproductive lag phase may be shortened by increasing the inoculum size and ensuring that the growth environment (medium, pH, temperature, etc.) in the bioreactor is the same as the one in which the inoculum was grown. Typically, the inoculum should be in the late exponential phase of growth. The volume of a microbial inoculum should between 5 and 10 percent of the volume of the medium being inoculated. Larger inocula are needed for slower growing cells such as animal and plant cells. In cultivation of animal cells, the inoculum size is selected to provide an initial cell count of (2–4) × 105 cells/ml. After the lag period, the exponential growth persists usually until an essential nutrient (the growth limiting nutrient or substrate) runs low in the medium, or until some inhibitory product of metabolism accumulates to a growth inhibitory concentration. The cells then enter a phase of zero net growth, or the stationary phase (Fig. 29). This is followed by a decline phase or death phase in which there is a net loss of biomass as cells die and lyse. Usually an attempt is made to achieve a maximum growth rate and maintain it for as long as possible. The biomass growth rate in a bioreactor, i.e., the change in biomass concentration with time, or dX/dt, depends on the viable biomass concentration X present at any time. In other words, growth is self catalyzing, or autocatalytic. In exponential growth, the growth rate is expressed as follows:

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FIGURE 29 A typical biomass growth profile in batch culture.

dX = µX , dt

where µ is a constant known as the specific growth rate. Equation (3) may be written in its integrated form, as follows: ln

X = µt , Xo

(4)

where X o is the initial biomass concentration at any time t = 0 and X is the concentration at time t. The time to double the biomass concentration, or the doubling time td , may be estimated from Eq. (4) by substituting td for t and 2X o for X o ; thus, td =

µ=

(3)

ln 2 . µ

(5)

Some typical values of the doubling time are noted in Table I for the various kinds of microbial and other cells. In steady-state continuous culture in which the cells experience an unchanging environment, the specific growth rate depends on the concentration S of a growth limiting substrate. This dependence is generally described by Monod kinetics, as follows: TABLE I Typical Doubling Times Cell type

t d (min)

Bacteria Yeasts Molds Protozoa Hybridomas Plant cells

20–45 90 160 360 630–1260 3600–6600

µmax S , Ks + S

(6)

where µmax is the maximum specific growth rate and K s is the value of S at which the specific growth rate is half of its maximum value. K s is known as the saturation constant. The specific growth rate increases with increasing substrate concentration in a hyperbolic manner, as shown by the solid line in Fig. 30. The figure also clarifies the meanings of µmax and K s . Sometimes, the growth rate is suppressed in the presence of too much substrate and growth is said to be substrate inhibited. In substrate inhibited culture, the specific growth rate attains a maximum value as the substrate concentration is increased and then the growth rate declines, as shown by the dashed line in Fig. 30. Note that in Fig. 30, the µmax values are different for the two growth profiles shown. The substrate inhibited growth may be described by the following equation: µ=

µmax S S2 Ks + S + Ki

,

(7)

where K i is the inhibition constant. In other cases, the growth rate may be subject to inhibition by a product of metabolism. 2. Productivity Productivity of a bioreactor is the quantity of product produced per unit volume in unit time. For a batch bioreactor with the growth profile shown in Fig. 29, the biomass productivity P at any time t is the slope of the straight line

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FIGURE 30 Dependence of the specific growth rate µ on the concentration S of the growth limiting substrate.

joining the biomass concentrations at t = t and t = 0 in Fig. 31; i.e., P=

X . t

(8)

This definition of productivity disregards any time invested in preparing the fermenter prior to inoculation of the batch. Lower values of productivity result when the additional preparatory time is taken into account. Often, the product of a fermenter is not the biomass per se, but a compound produced by the cells. Examples of such products are antibiotics and monoclonal antibodies. The kinetics of product formation may mirror those of the cell growth, or they may be quite different. Products known as secondary

metabolites (i.e., ones that are nonessential to the cell) are often produced after the growth has ceased. An important operational variable for continuous flow bioreactors is the dilution rate D, or the volume flow rate of the feed medium divided by the constant volume of the broth in the reactor. The biomass productivity of a continuous culture is simply the dilution rate D multiplied by the biomass concentration X in the harvest stream. In a continuous flow well-mixed bioreactor, the biomass concentration varies with dilution rate, as shown in Fig. 32. Also, the productivity increases with increasing dilution rate until the dilution rate approaches close to the maximum specific growth rate (Fig. 32). Any further increase in dilution rate causes a sharp decline in productivity and

FIGURE 31 Biomass productivity in batch culture.

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FIGURE 32 Variation of the steady sate biomass concentration and the productivity with dilution rate in a well-mixed continuous flow bioreactor.

washout of cells from the bioreactor. In practice, the dilution rate must remain quite a bit less than the value needed for optimal productivity (Fig. 32), or cells may be washed out because of an inadvertent slight increase in the dilution rate. If viable biomass from the outflow of a continuous flow well-mixed bioreactor is recycled to the reactor, as in Fig. 23b, then the reactor may be operated at a dilution rate greater than the maximum specific growth rate. Such a bioreactor provides a greater biomass productivity compared to one that does not recycle the biomass and the biomass concentration in the reactor outflow stream is also greater.

Instead of viable cells, a bioreactor may use nonviable cells and isolated enzymes as the biocatalyst. The reaction in such a bioreactor may obey Michaelis-Menten kinetics but other kinetic patterns are also observed. For a reaction that obeys Michaelis-Menten kinetics, the rate of reaction (i.e., the rate of consumption of the substrate, −d S/dt) depends on the concentrations of the substrate S and the enzyme e, as follows: dS kr eS = , dt Km + S

(9)

where kr is the rate constant and K m is known as the Michaelis-Menten constant. The dynamics of a bioreactor are often analyzed in terms of the conversion Cs of the substrate, where Cs is the fraction of the original substrate transformed to a product, i.e., Cs =

kr Et = So Cs − K m ln(1 − Cs ) , VL

So − S . So

(10)

(11)

where E is the total amount of enzyme in a bioreactor of volume VL . When the reaction is carried out in a continuous flow well-mixed bioreactor, the expression for the conversion is as follows: kr E K m Cs = So Cs + , F 1 − Cs

3. Enzyme Kinetics

−

In Eq. (10), So is the initial concentration of the substrate in the reactor and S is the concentration at time t. For Michaelis-Menten kinetics in a well-mixed batch bioreactor, the conversion of the substrate at any time t is governed by the relationship:

(12)

where F is the volume flow rate of the feed. Similarly, in a packed bed bioreactor, the expression for the conversion is the following: kr E = So Cs + K m ln(1 − Cs ) . F

(13)

Because F is the volume processed in time t in a continuous flow bioreactor and VL is the corresponding volume in a batch reactor, a comparison of Eqs. (11) and (13) shows that batch and plug flow (i.e., packed bed) bioreactors containing the same amount of enzyme will achieve equal conversions in a given time. This is a general conclusion, irrespective of the reaction kinetics. A continuos flow packed bed enzyme bioreactor may be advantageous relative to batch reactor, as the unproductive time for batch preparation could be eliminated in the continuous flow unit. However, the batch reactor may have other important advantages such as the ease of pH control in a well-mixed device.

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When the substrate concentration S is much greater than K m , Eqs. (12) and (13) reduce to the same form. In this case, the continuous flow stirred reactor and the plug flow device achieve similar conversion values in a given time. In contrast, when S K m , the reaction rate becomes first order in the substrate concentration (see Eq. (9)), and the plug flow reactor provides higher conversion values in comparison with the well-mixed continuous flow device. In the latter bioreactor, all the enzyme would be exposed to the same low concentration of the substrate which is not useful except when the reaction is inhibited by the substrate.

IV. CONCLUDING REMARKS A bioreactor is an indispensable part of any bioprocess irrespective of whether the process degrades pollutants or produces substances such as foods, feeds, chemicals and pharmaceuticals, and tissues and organs for use in biomedicine. The variety of bioprocesses is tremendous and many different designs of bioreactors have been developed to meet the different needs. In all cases, the bioreactor must provide the environmental conditions necessary for the culture. The specific demands are often conflicting and achieving optimal performance requires attaining the proper balance among the different requirements. Success of a bioprocess depends critically on good design and operation of the bioreactor.

SEE ALSO THE FOLLOWING ARTICLES BIOENERGETICS • BIOMASS, BIOENGINEERING OF • ENZYME MECHANISMS • MAMMALIAN CELL CULTURE

• METABOLIC ENGINEERING • PHOTOCHEMISTRY, MOLECULAR • SEPARATION AND PURIFICATION OF BIOCHEMICALS

BIBLIOGRAPHY Atkinson, B. (1974). “Biological Reactors,” Pion Press, London. Bailey, J. E., and Ollis, D. F. (1986). “Biochemical Engineering Fundamentals,” 2nd Ed., McGraw-Hill, New York. Chisti, Y. (1989). “Airlift Bioreactors,” Elsevier, London. Chisti, Y. (1999). Solid substrate fermentations, enzyme production, food enrichment. In “Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis, and Bioseparation,” (M. C. Flickinger and S. W. Drew, eds.), Vol. 5, pp. 2446–2462, Wiley, New York. Chisti, Y. (1999). Modern systems of plant cleaning. In “Encyclopedia of Food Microbiology” (R. Robinson, C. Batt, and P. Patel, eds.), pp. 1806–1815, Academic Press, London. Chisti, Y., and Moo-Young, M. (1999). Fermentation technology, bioprocessing, scale-up and manufacture. In “Biotechnology: The Science and the Business” (V. Moses, R. E. Cape, and D. G. Springham, eds.), 2nd ed., pp. 177–222, Harwood Academic Publishers, New York. Deckwer, W. D. (1992). “Bubble Column Reactors,” Wiley, New York. Doran, P. M. (1999). “Design of mixing systems for plant cell suspensions in stirred reactors,” Biotechnol. Prog. 15, 319–335. Moser, A. (1981). “Bioprocess Technology,” Springer-Verlag, New York. Nienow, A. W. (1998). “Hydrodynamics of stirred bioreactors,” Appl. Mech. Rev. 51, 3–32. Tredici, M. R. (1999). Bioreactors, photo. In “Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis, and Bioseparation” (M. C. Flickinger and S. W. Drew, eds.), Vol. 1, pp. 395–419, Wiley, New York. Van’t Riet, K., and Tramper, J. (1991). “Basic Bioreactor Design,” Dekker, New York. Varley, J., and Birch, J. (1999). “Reactor design for large scale suspension animal cell culture,” Cytotechnology 29, 177–205. Willaert, R. G., Baron, G. V., and De Backer, L. (eds.) (1996). “Immobilised Living Cell Systems: Modelling and Experimental Methods,” Wiley, Chichester.

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Fiber-Optic Chemical Sensors David R. Walt Israel Biran Tarun K. Mandal Tufts University

I. Introduction II. Fundamental Principles of Fiber-Optic Chemical Sensors III. Sensing Schemes for Fiber-Optic Chemical Sensors IV. Applications of Fiber-Optic Chemical Sensors V. Recent Developments VI. Conclusions

GLOSSARY Absorption Process by which electromagnetic energy is transferred to an atom or molecule. Different molecules absorb light at different discrete wavelengths and therefore each molecule has a characteristic absorption spectrum. Antibody Biologically derived molecule able specifically to recognize and bind to another molecule (antigen). Biosensor Sensor in which the sensing material is of biological origin (e.g., enzyme, antibody, cell, DNA). Extrinsic sensor Sensor based on indirect analysis using an indicator (compound that changes optical properties by reaction with an analyte). Fluorescence Emission of light at a longer wavelength by molecules or other chemical species after excitation at a shorter wavelength.

Imaging fiber Bundle of coherently ordered individual optical fibers through which an image can be transmitted from one side to the other. Immobilization Attachment of chemical species to surfaces, polymers, or other insoluble substrates. For example, with optical sensors, indicators are attached to the optical fiber surface. Indicator Molecule that undergoes changes in optical properties on interaction with chemical species. Intrinsic sensor Sensor based on the direct spectroscopic detection of a molecule of interest. Optical fiber Transparent filament made out of glass or plastic through which light can propagate by total internal reflection. Sensing element Combination of sensing materials (indicators and/or biological compounds) and a transducer (optical fiber). Sensing material Material (indicator, dye, polymer,

803

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804 biological molecule) that is immobilized to the optical fiber surface and can change its physicochemical properties upon interaction with an analyte. Total internal reflection Reflection of light at the interface between two materials of different refractive index. In optical fibers, the core refractive index is higher than the clad refractive index. When light is introduced into the core, it is reflected at the core–clad interface.

A CHEMICAL SENSOR is an analytical device that can measure the concentration of a specific chemical or a group of chemicals in a sample of interest. The basic structure of a chemical sensor includes (a) a sensing material that selectively interacts with the analyte and (b) a transducer (e.g., electrochemical, optical, thermal, or mass) that can transform this interaction into a measurable signal. This signal should be proportional to the magnitude of the changes in the physicochemical properties associated with the interaction between the sensing material and the analyte. Ideally, chemical sensors should operate in a continuous and reversible manner. Fiber-optic chemical sensors are analytical devices incorporating optical fibers as part as their optical transducing system. Optical fibers are small and flexible “wires” made out of glass or plastic that can transmit light signals, with minimal loss, for long distances. The signals generated by the sensing materials, which are usually immobilized to the fiber surface, are transmitted through the optical fibers and can be measured by using different optical methods such as absorption, fluorescence, and Raman spectroscopy. Fiber-optic chemical sensors can be used for remote analytical measurements in applications including clinical, environmental, and industrial process monitoring. Several fiber-optic chemical sensors are commercially available and it is expected that recent developments in optical technologies and the research efforts to use these technologies for fiber-optic sensor development will lead to a number of commercially available fiber-optic chemical sensors for various applications.

I. INTRODUCTION Fiber-optic chemical sensors are composed of a sensing material and a transducer. The transducer converts the recognition and sensing events obtained by the sensing materials into a response such as an optical signal. Optical measurements can provide rapid, sensitive, and nondestructive analysis of many important compounds. In fiberoptic chemical sensors, optical fibers are used to transmit the optical signal to the measurement device, enabling a remote detection of the analyte in the sample. The use

Fiber-Optic Chemical Sensors

of optical fibers for sensing applications was first suggested in the mid-1960s. Since then, various fiber-optic chemical sensor types have been developed for detecting numerous analytes. In the last decade, due to the rapidly growing use of fiber optics for telecommunication applications, new fiber-optic technologies have been developed resulting in high-quality and inexpensive optical fibers that can be used for chemical sensing applications. Optical fibers are remarkably strong, flexible, and durable. These features and their nonelectrical nature make them highly suitable for different industrial and environmental applications where safe sensing in hazardous and harsh environments is needed (e.g., monitoring of chemicals in nuclear plants or toxic gases in petrochemical plants). Fiber-optic chemical sensors are also widely used in the clinical field since their small dimensions allow them to be used for in vivo sensing thereby eliminating the need to procure samples. Furthermore, as described later in Section IV, fiber-optic chemical sensors can be incorporated into optical fiber bundles used for in vivo imaging (endoscopes) to provide both analytical information and imaging capabilities. One significant advantage of employing optical fibers is that multiple optical signals can be transmitted and measured simultaneously, thereby offering multiplexing capabilities. Optical fibers and the instrumentation used in fiberoptic chemical sensors are described in Section II. We describe how different optical phenomena, generated by different sensing mechanisms, can be applied to optical fibers to measure analytical signals. These sensing mechanisms are described in Section III. Section IV reviews several fiber-optic chemical sensor analytical applications in the clinical, industrial, and environmental fields and Section V reviews recent developments in fiber-optic chemical sensors.

II. FUNDAMENTAL PRINCIPLES OF FIBER-OPTIC CHEMICAL SENSORS The basic design of a fiber-optic chemical sensor system is shown in Fig. 1. The fiber-optic chemical sensor’s main components are (a) a light source, (b) optical fibers to both transmit the light and act as the substrate for (c) the sensing material, and (d) a detector to measure the output light signal. Usually computers or microprocessors control the fiber-optic chemical sensor instrumentation and are employed to analyze the output signals. In this section, optical fibers and their basic characteristics are described. Fiber-optic chemical sensor instrumentation and the optical phenomena employed are also described.

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FIGURE 1 Schematic diagram of a fiber-optic chemical sensor system with examples of environmental, clinical, and industrial applications.

A. Optical Fibers 1. Basic Characteristics Optical fibers are waveguides made out of glass or plastic, through which light can be transmitted. Optical fibers transmit light very efficiently, which is why they are so useful for many applications. The light transmission through the fiber is based on the phenomenon of total internal reflection (TIR). Optical fibers consist of a core with a refractive index n 1 surrounded by a cladding with a lower refractive index n 2 (Fig. 2). The difference between the refractive indices enables the core–clad interface to effectively act as a mirror such that a series of internal reflections transmits the light from one end of the fiber to the other as shown in Fig. 3a. Several principles related to the light transmission through the optical fiber are significant for fiber-optic chemical sensor function and design: 1. The Critical Angle. If light strikes the cladding at an angle greater than the critical angle ϕc , the light is totally internally reflected at the core–clad interface (Fig. 3a). If light strikes the cladding at an angle less than the critical angle, as shown in Fig. 3b, it is partly reflected and partly

refracted. The critical angle is defined by the ratio between the clad and the core refractive indices, sin ϕc = n 2 /n 1 .

(1)

2. Acceptance Cone. In order to get high light transmission, light should propagate through the fiber by a series of total internal reflections. This transmission is achieved if the angles of the light entering the fiber are within the acceptance cone as shown in Fig. 3c. The acceptance cone

FIGURE 2 Schematic diagram of an optical fiber showing core and clad structure.

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FIGURE 3 Propagation of light through the optical fiber occurs when the total internal reflection condition exists at the interface between the core (index of refraction n1 ) and cladding (n 2 ) such that n1 > n 2 . (a) Light entering the fiber is totally internally reflected (TIR) if the light angle is greater than the critical angle ϕc . (b) Light will be partly reflected and partly refracted if the light angle is less then the critical angle ϕc . (c) Light will propagate in TIR only when the entering light angle is within the acceptance angle.

size depends on the refractive indices of the core and the clad and also on the refractive index of air n 0 ,   n 21 − n 22 sin ϕm = . (2) n0 3. Numerical Aperture. The acceptance cone’s width determines the efficiency of light collection of the fiber and can also be described in terms of the numerical aperture (NA), NA = n 0 sin ϕm .

(3)

A high NA indicates a wide acceptance cone and better light-gathering capabilities of the fiber. A typical NA value for a high-quality glass fiber is 0.55 but fiber NAs as high as 0.66 or as low as 0.22 have been used for sensing. 2. Optical Fiber Types Optical fibers are usually made of glass in a process that involves heating a glass preform and then drawing the

fiber from the melted hot glass. In most fiber-optic chemical sensors, glass fibers are used to transmit light in the visible and near-infrared regions of the optical spectrum (400 < λ < 700 nm). In some cases, when the light employed is in the UV region, quartz (pure silica) fibers are used as the core material and doped silica (with lower refractive index) is employed as the cladding material. Optical fibers made of silver halide or chalcogenides are used to transmit light in the infrared region of the spectrum (λ > 700 nm). Plastic fibers are also used for fiber-optic chemical sensors; these fibers are very flexible and cheap but their optical characteristics are inferior to those of glass fibers and their heat tolerance is lower. 3. Optical Fiber Configurations Optical fibers are produced in many different configurations, formats, and sizes. For some fiber-optic chemical sensor applications, single optical fibers with diameters ranging from 50 to 500 µm are employed. For

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FIGURE 4 Optical fiber bundle fabrication and use for imaging. (a) Fiber bundles are constructed from thousands of individual single fibers that are fused together. (b) Coherent bundles can be used for imaging.

other fiber-optic chemical sensor applications, fiber-optic bundles comprising thousands of identical single fibers (each with a diameter of a few micrometers) are employed, as shown in Fig. 4a. The fibers can be bundled in a coherent or random fashion and used to enhance light transmission. In coherent fiber bundles, the position of each fiber on one end is identical to its position on the other end. These coherent fiber bundles are mostly used for imaging applications as shown in Fig. 4b. The coherent fibers allow each individual fiber to transmit light in an addressable manner from one end of the bundle to the other. In some recent applications, these bundles are used for imaging and sensing simultaneously.

requirements for the application, for example, the use of nontoxic agents and biocompatible materials for clinical applications. There are two basic configurations for fiberoptic chemical sensors: (a) A single fiber is used to transmit the light from the light source to the sample region and back to the detector, as shown in Fig. 1. (b) Multiple fibers are used, one to transmit the light to the sample region and the rest to transmit light from the sample region to the detector, as shown in Figs. 5a and 5b. For the second configuration, the most common format is a bifurcated fiber. Bifurcated fibers are fabricated by fusing two fibers together on one end, leaving the other ends free. The fused side is used for the sensing and the other ends of the fiber are connected to the light source and to the detector, as shown in Fig. 5c.

B. Fiber-Optic Chemical Sensor Design and Instrumentation Each fiber-optic chemical sensor system design depends intimately on the nature of the analytes detected, the particular conditions at the measurement site, and the specific

1. Light Sources The light sources used for fiber-optic chemical sensors should provide sufficient light intensity within the sensor

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FIGURE 5 Design principle of fiber-optic chemical sensors. (a) Two single fibers. (b) Two fiber bundles. (c) Bifurcated fiber.

wavelength operating range. In addition, the light output should be stable over long time periods since light fluctuations may add noise to the measurement and reduce the sensor sensitivity. Tungsten–halogen lamps are often used as a light source to provide high-intensity light and a continuous emission spectrum over wavelengths ranging from 300 to 2700 nm. This lamp is very useful because of its small size and high stability, and the potential to use it in portable devices. Light-emitting diodes (LEDs) are also used as light sources for fiber-optic chemical sensors because they offer spectral consistency and small size; however, they are limited in their light intensity. Lasers are an important light source for fiber-optic chemical sensors since they supply very intense monochromatic light. Lasers are used for fluorescence-based fiber-optic chemical sensors in which fluorophores with low quantum yields are used. 2. Optical Signal Detectors In most fiber-optic chemical sensor systems, the light transmitted from the sensing element (output light) is measured by using photon detection devices, which absorb photons and convert them into electrical signals. Several photon detectors are available. The simplest and cheapest detector is the silicon photodiode. In this solid-state device, photon detection is based on p–n semiconductor

Fiber-Optic Chemical Sensors

junctions. When photons strike the p junction, electrons move into the conducting band and act as charge carriers. This process generates a current that is proportional to the incident light intensity. A series of photodiodes can be arranged in a linear array and used to detect the entire spectrum simultaneously. These devices offer advantages such as small size, wide spectral range, and low power requirement; however, photodiode sensitivity is relatively low. Photomultiplier tubes (PMT) are more sensitive than photodiodes and are used for low-light-level detection. Photons that strike the photocathode cause the ejection of photoelectrons that are attracted to the photomultiplier electrodes (also called dynodes). The dynodes are arranged such that each dynode is held at a higher potential than the previous one. When a high-energy photoelectron strikes the first dynode, it causes the ejection of several photoelectrons from the dynode surface. These cascade processes continue as more photoelectrons strike each subsequent dynode, resulting in a high amplification of the initial photon. In addition to their high sensitivity, PMTs have high signal-to-noise ratios and respond very fast. PMT detectors, however, are temperature sensitive and can be irreversibly damaged if exposed to high light levels. In some fiber-optic chemical sensors, the optical signal measurement involves image acquisition and analysis. Charge-coupled device (CCD) chips are typically used to capture the output image. The CCD is a twodimensional integrated-circuit chip array made up of several layers of silicon and an array of gate electrodes. These electrodes, often referred as metal-oxide semiconductors (MOS), are individually connected to an external voltage source. When photons strike the chip’s light-sensitive silicon layer, they create electrical charges that are detected by the gate electrodes. This detection process is called charge coupling and involves the transfer of voltage between the two gate electrodes. Since each pair of gate electrodes can detect the localized photon strikes, intensity and spatial position of the image are simultaneously measured. CCD chips offer additional advantages including their small size (a few square millimeters to a few square centimeters), high sensitivity, and stability. The limitations of CCD chips are the requirement for low-temperature operation and their relatively high cost.

C. Optical Phenomena Employed for Sensing in Fiber-Optic Chemical Sensors Chemical sensing via optical fibers is performed by monitoring the changes in selected optical properties. Interactions of light with the analytes are measured either directly or by using indicators. The light is launched from a light source into the fiber and guided to the measurement site. The absorbed, reflected, scattered, or emitted light

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can be measured using several different optical phenomena. These phenomena transduce the interactions of light with the sensing materials into an (ideally) quantitative signal that can be correlated to the analyte identities and concentrations. 1. Absorption Absorption is based on the light intensity changes due to modulation by a substance in the sample. Light absorption is a process in which electromagnetic energy is transferred to an atom or a molecule. This energy promotes the transition of the molecule from the ground energy state to a higher energy excited state. The resulting energy is dissipated nonradiatively (i.e., thermally) to the medium when the excited state relaxes to the ground state. Each molecule (analyte) can be excited at a single wavelength or several wavelengths, which furnishes a unique absorption spectrum characteristic of the molecule. The absorbance changes are related to the analyte concentration [C] via the Beer–Lambert relationship: A = log(I0 /I ) = ε · [C] · l,

(4)

where A is the optical absorbance, I0 and I are the intensities of transmitted light in the absence and presence of the absorbing species, respectively, l is the effective path length, and ε is the molar absorption coefficient. In practice, optical fibers are connected to a spectrophotometer and the measured changes correlate the analyte concentration to the absorption at a given wavelength.

FIGURE 6 Typical fluorescence spectrum showing the strokes shift at longer wavelengths from the excitation spectrum.

3. Time-Resolved Fluorescence Spectroscopy This method is based on the excited-state lifetime. The light intensity emitted from molecules excited by a short pulse of light decays exponentially with time. This decay pattern is unique for each molecule and can be used for analytical purposes. Alternatively, a phase shift method can be employed to measure the fluorescence lifetime. A sinusoidally varying excitation light source is used and the phase shift between the excitation waveform and the emission waveform can be used to detect the analytical signal. 4. Fluorescence Energy Transfer

2. Fluorescence When fluorescent molecules are excited at a specific wavelength, the molecule reemits radiation at a lower energy, i.e., a longer wavelength. The absorption of the excitation light shifts the molecule’s energy from the ground state to a higher energy state. The molecule emits fluorescent light when it returns to the ground state. The distinct ranges of wavelengths over which the molecule is excited and emits are well defined and simple to detect, as shown in a typical spectrum of a fluorescent molecule in Fig. 6. Concentrations of the fluorescent analytes are measured by transmitting an excitation light through the optical fiber and measuring the light emission intensity using a detector. A nonfluorescent analyte can be measured indirectly if its interaction with an indicator molecule changes the indicator emission intensity (see Section III.B). A decrease in fluorescent intensity due to fluorescence quenching can also be used for sensing. In this case, the analyte’s interaction with a fluorescent molecule causes a decrease in fluorescence (quenching). The magnitude of the fluorescence decrease is related to the analyte concentration.

This phenomenon occurs when two distinct fluorophores are present. If the emission spectrum of one fluorophore overlaps with the excitation spectrum of a second fluorophore and the two fluorophores are in sufficient prox˚ then the excited fluorophore (donor) can imity (<100 A), transfer energy nonradiatively to the second fluorophore (acceptor). This transfer results in an increase in light emission by the acceptor and a decrease in light emission from the donor. When an energy transfer pair of fluorophores is used to label two molecules that can interact (antibody–antigen, enzyme–substrate), they can be use for sensing in fiber-optic chemical sensors. 5. Raman Spectroscopy In Raman spectroscopy, light is scattered from the molecule in different directions and is shifted to both higher and lower frequencies. The shift in magnitude is equal to the characteristic vibration frequencies of the molecule, resulting in a unique spectrum for each molecule. Optical fibers are used as light guides for Raman spectroscopy because the optimum wavelengths for the

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FIGURE 7 Schematic principle of surface plasmon resonance (SPR) sensing. (a) Light is absorbed only at the resonant angle R . (b) Analyte binding shifts the light resonant angle to angle R1 .

analysis of many different analytes are in the visible or near-IR range. The limitation to the Raman method is the low intensity of the scattered light. A significant enhancement of the scattered light signal (106 ) is observed when the analyte molecules are adsorbed on metal surfaces. In this method, called surface-enhanced Raman scattering (SERS), two optical fibers are typically used. One fiber is used to transmit the excitation light to the sensing area (glass slide covered with a metal layer, usually silver) and the second fiber is used to collect the scattered radiation.

6. Surface Plasmon Resonance (SPR) Very small changes in the refractive index on the sensor surface can be measured using SPR, including changes caused by the binding of molecules. SPR occurs at the surface of a thin metal layer placed on a glass slide or other dielectric material such as an optical fiber. Incident light with a specific angle is resonantly coupled to the surface plasmons (oscillating electrons at the edges of the metal) in the metal layer because of their matched frequencies.

Energy is absorbed in this resonance, resulting in a sharp decrease in the reflected light (Fig. 7). This phenomenon is used for analytical applications since any changes at the metal layer surface, i.e., binding of analyte, cause a shift in the resonant angle. The magnitude of the shift from the initial resonant angle before the binding of the analyte is proportional to the concentration of the bound analyte.

7. Evanescent Wave Spectroscopy When light propagates by total internal reflection (TIR) through an optical fiber or a planar optical waveguide, a small portion of the electromagnetic wave penetrates the region of lower refractive index, thereby generating an evanescent field that exponentially decays as a function of the distance from the fiber or waveguide surface (Fig. 8a). When a small part of the fiber cladding is removed, the evanescent field around the exposed core can be used for sensing at the core–air or core–liquid interface. Binding of molecules to the exposed region may induce several measurable changes in the properties of the light transmitted

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FIGURE 8 Schematic principle of evanescent wave field sensing. (a) The evanescent field is formed when a small portion of the cladding is removed. (b) The bound analytes absorb some of the light propagating through the fiber. (c) Excitation light (λex ) transmitted through the fiber excites the bound fluorescent molecules and emitted light (λem ) is measured at the fiber output.

through the fiber. In one example, the target molecules absorb some of the light and reduce the transmitted light intensity, as shown in Fig. 8b. Alternatively, the binding of fluorescent molecules can be determined by employing light that corresponds to the molecule’s excitation wavelength and measuring the emitted light, as shown in Fig. 8c.

III. SENSING SCHEMES FOR FIBER-OPTIC CHEMICAL SENSORS Fiber-optic chemical sensors can be divided into two categories based on their structure: (a) Intrinsic sensors are based on the analyte’s intrinsic optical properties, and (b) extrinsic sensors are based on sensing materials (chemical or biological) immobilized to the fiber surface, with

the optical fibers used only as a transmission pathway between the sensing materials and a remote measurement device. The basic concepts of these two sensing schemes are shown in Fig. 9. Optical fibers allow measurements to be made directly in the sample. Intrinsic sensors usually have a simple structure and a fast response time, but they are not as selective as extrinsic sensors because many groups of compounds exhibit similar optical properties when measured directly. Extrinsic sensors provide an additional level of selectivity. In this section, we describe both sensor types and their use for fiber-optic chemical sensors. A. Intrinsic Sensing Mechanism—Direct Spectroscopy Many different spectroscopic techniques are used for direct spectroscopic measurements. These techniques

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FIGURE 9 Two basic fiber-optic chemical sensing schemes: (a) Intrinsic, (b) extrinsic.

include transmission or absorption, attenuated total reflection, photoacoustic, fluorescence, light scattering, infrared, and Raman spectroscopy. In this section, we describe how some of these techniques are employed for use with optical fibers to measure the concentration of chemical species either in the liquid phase or the gas phase. Generally, an optical fiber is coupled with an instrument such as a spectrophotometer or fluorimeter. The particular optical fibers used are determined by the specific requirements of each type of spectroscopic technique. 1. Liquid-Phase Sensing Transmission spectroscopy has found wide application in chemical, biological, and environmental monitoring because of its intrinsic safety and ease of application. This method generally is used to monitor a single analyte in a harsh analytical medium. Transmission spectroscopy is also used for simultaneous detection of several species at trace levels in a complex medium. Light is focused into the fiber, passes through the medium, and then is coupled through the return fiber into the input slit of the spectrom-

eter. The light losses due to the small core diameter and acceptance angle of the optical fiber are reduced by attaching a convergent lens at the remote sensor head (Fig. 10). This lens causes the light from the illumination fiber to pass through the sensing region and refocuses the transmitted light into the return fiber. Using this simple technique with an optical fiber allows one to measure the absorbance of the analyte directly. For example, the salt concentration in an electroplating bath can be easily measured by monitoring the solution absorbance. This technique is also used for in vivo analysis of components in human blood (see Section IV.A). Optical fibers are commonly used for remote monitoring of fluorescent analytes. Fiber-optic chemical sensors can provide both qualitative and quantitative information about the analyte under consideration. Since each analyte has different fluorescent properties, selective measurements can be performed by choosing the correct excitation and emission wavelengths. The fluorescence bands are usually quite broad and the bands for a class of compounds overlap, so it may be difficult to distinguish among them. Fluorescence lifetime measurements are sometimes

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FIGURE 10 A typical arrangement for remote measurement of sample absorption using optical fibers.

used in such cases, as they provide an additional parameter for distinguishing such compounds. For example, phenol, toluene, and xylene are single-ring aromatic compounds with similar emission peaks. It is not possible to distinguish between them by simply measuring their fluorescence spectra. It is possible, however, to distinguish between them by measuring their fluorescence decay times. Also, low concentrations of dissolved polyaromatic hydrocarbons, such as benzene, naphthalene, and pyrene, in natural water can be determined selectively by measuring the fluorescence lifetimes of the individual molecules. The different techniques for performing fluorescence lifetime measurements are described in Section II.C.3. Remote fiber-optic Raman spectroscopy has found applications in process control. The principal advantage of this method is that many different molecules can be detected directly in solids, liquids, or gases, in complex media, or in harsh environments (i.e., high temperature or pressure). This technique is most sensitive to compounds that are IR-active. A large amount of qualitative and quantitative information can be gathered from the Raman spectrum without using any indicator chemistry. The low intensity of the Raman-scattered light is the major weakness of this technique. In addition, organic molecule fluorescence usually interferes with Raman spectra. This technique has been widely used for in situ monitoring of chemical reactions under harsh conditions. One application of Raman spectroscopy using optical fibers is to monitor epoxycuring reactions. The extent of curing and the system tem-

perature can be measured simultaneously. The degree of epoxy curing is calculated by taking the ratio of the epoxide ring stretch at 1240 cm−1 , which is linearly dependent on the progress of the curing reaction, and the 1186-cm−1 vibration of a fragment not affected by the cure. Raman spectroscopy with optical fibers is also used for on-line monitoring of water in sodium nitrate slurries in the nuclear industry. The Raman method is a nondestructive optical technique that can also provide detailed information about the molecular composition of tissues and it recently has been used for in vivo determination of the molecular composition of an arterial wall. 2. Gas-Phase Sensing The most common fiber-optic gas sensor is based on spectral transmission analysis. Such analysis is commonly performed using two wavelength regions: 250–500 nm and ca. 1–8 µm. Absorption or emission in the lower wavelength region corresponds to electronic transitions within atoms or molecules, and is a useful region for measuring the energy changes associated with the transitions of a large number of gaseous species. The longer wavelengths cover the near- and mid-IR regions of the spectrum, and contain information about vibrational absorption bands of the gases. Conventional silica fibers are good for measuring the near-IR absorption lines but they are not good at the lower or mid-IR regions due to high attenuation and an increase in opacity of the silica, respectively. Therefore, a

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FIGURE 11 A fiber-optic pH sensor employing reversible protonation of dye (Dy− ).

long-path or multipass cell and a well-designed optoelectronic system are required reliably to detect low levels of gases. Although IR fibers can be used, they are fragile and expensive compared to conventional silica fibers. Direct absorption methods are mostly used to monitor low concentrations of gases such as nitrogen dioxide, methane, and hydrogen.

B. Extrinsic Sensing Mechanism—Indicator Chemistry Extrinsic fiber-optic chemical sensors are constructed by immobilizing indicator chemistries on the fiber tip or on the annulus of the fiber. In this section, the mechanism of the chemical and biological reagents employed for sensing are described as well as methods for immobilizing sensing materials.

1. Chemical Sensing Reagent—Optrodes a. pH sensing. Among all types of sensors, the pH sensor is the most widely developed and has received the most attention because of the importance of pH measurements in scientific research as well as various physiological, environmental, and industrial processes. Fiber-optic pH sensors offer several advantages compared to standard glass electrodes for pH measurement, including their immunity from electromagnetic interference, their small size, their ability to perform remote sensing, and their application to both in vivo and in situ measurements. Optical sensors also possess some disadvantages, such as photobleaching of the dye, leaching of dye from the immobilized surface, and interference by ambient light. The pH measurement is based on pH-dependent changes of the optical properties of an indicator dye immobilized on the fiberoptic surface. The indicator reversibly reacts with protons,

and as a result, the absorption, reflection, or fluorescence properties of the indicator change. A schematic of a typical pH sensor is shown in Fig. 11. Some common dyes used for pH sensors are listed in Table I. The fiber-optic, absorbance-based pH sensor monitors the absorbance change of the immobilized dye as a function of the pH change of the analytical medium. The Beer– Lambert law can be applied to an absorbance based pH sensor. The concentration of the dye [Dy] is related to the absorbance according to the following equation: A = log(I0 /I ) = ε [Dy] l,

(5)

where I0 and I are the intensity of transmitted light in the absence and presence of dye, respectively, l is the effective path length, and ε is the molar absorption coefficient. Usually the concentration of the base form of a weak acid indicator is measured, which is a measure of the pH-dependent degree of ionization of the indicator. The dissociation of a dye and the acidity constant K a are expressed in the following equations, respectively: Dy − H = Dy− + H+ ,

(6)

[Dy− ][H+ ] . [Dy − H]

(7)

Ka =

Combining the equilibrium expression for the acid dissociation of a dye with the expressions for pH and pK a gives Measured absorbance (A) Absorbance of the total dye in base form =

1 10(pKa −pH) + 1

(8)

This relationship results in a sigmoidal plot of absorbance versus pH centered on the pKa value. In comparison to fluorescence, the absorbance method is simple

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of the instrument and sensor. When I0 is constant, Eq. (9) can be simplified to

TABLE I Common Indicators Indicator a

pKa

I F = k[Dy],

Absorbance-based indicators Bromothymol blue Chlorophenol red Dibromo-xylenol blue Neutral red Nitrazine yellow Palatine chrome black Phenol red Phenoltetrachloro-sulfonaphthalein

6.8 6.3 7.6 7.4, 5.9 6.5 7.4 7.6 7.0

Long-wave-absorbing pH indicators Methyl violet Malachite green Cresol red Bromophenol blue Naphtholbenzein Alizarin yellow R Alizarin Indigocarmine

0.0–1.6 0.2–1.8 1.0–2.0 2.8–4.8 8.2–10.0 10.0–12.0 11.0–12.4 11.4–13.0

Fluorescence indicators Fluorescein Eosin 2 ,7 -dichlorofluorescein 5(6)-carboxy-fluorescein Carboxy naphthofluorescein SNARF SNAFL

where k = k  I0 φ F εl. Again the concentration of the dye can be easily related to the pH of the solution. The measured fluorescence intensity can be represented by the same form of equation as shown in Eq. (8), Measured fluorescence Fluorescence of the total dye in base form =

2.2, 4.4, 6.7 3.25, 3.80 0.5, 3.5, 5.0 6.4 7.0 7.6 7.6, 7.3

a SNARF, seminaphthorhodafluor; SNAFL, seminaphthofluorescein.

and easy to use, but it is not very sensitive, requiring the use of a high concentration of pH indicator and a relatively thick sensing layer. Fluorescence-based fiber-optic pH sensors are more widely used due to their higher sensitivity. In this technique, the fluorescence intensity change of an immobilized dye is measured corresponding to a change in the medium pH. For example, the acid form of fluorescein does not fluoresce, but its conjugate base strongly fluoresces upon excitation. The concentration of deprotonated fluorescein is directly proportional to the measured fluorescence intensity and is dependent on the solution pH through its acid dissociation equilibrium. The intensity I F of fluorescence light returning from the sensor tip is proportional to the concentration of the dye in the sensor and the intensity of the exciting radiation I0 , I F = k  I0 φ F εl[Dy],

(10)

(9)

where l is the optical path length in the sensing layer, ε is the molar absorptivity, φ F is the quantum yield of fluorescence, and k  is a constant related to the configuration

1 10(pKa −pH)

+1

.

(11)

This relationship also results in a sigmoidal plot of intensity versus pH with a midpoint of the linear part of the curve corresponding to the pKa of the immobilized dye. A pH sensor prepared by immobilizing a particular dye is useful over approximately two pH units (±1 pKa ). Such a small pH range is a limitation of optical pH sensors compared to pH electrodes. A pH fiber-optic chemical sensor having a wide range of pH sensing capabilities can be constructed by immobilizing different dyes on a single fiber tip or a bundle of single-core fibers each containing a single dye with a particular pKa value. When several dyes each having a different pKa and a different optical spectrum are used, the pH in the region of each pKa is determined separately by measuring the change in the distinctive spectrum for that pH region. A fiber-optic pH sensor based on fluorescence energy transfer can be constructed by coimmobilizing a pH-sensitive fluorophore and a pH-sensitive absorber. For example, eosin (donor) and phenol red (acceptor) were coimmobilized in a polymer on the distal end of a silanized single-core optical fiber. Eosin’s emission spectrum overlaps with the absorption of the basic form of phenol red. The concentration of the basic form of phenol red increases with an increase in pH. As a result, energy transfer from eosin to phenol red increases and the fluorescence intensity of eosin decreases. Thus, the pH-dependent absorption change of phenol red can be detected as changes in the fluorescence signal of eosin. An evanescent-field-type pH sensor can be fabricated by replacing the cladding layer with a thin layer of pH-sensitive dye embedded in a polymer matrix. The basic designs are shown schematically in Fig. 8c. The measurement is based on the interaction of the evanescent wave with the dye in the coated cladding. A portion of the resulting dye fluorescence is coupled back into the fiber through the same mechanism that generates the original evanescent wave. Other than dye-based indicators, certain conducting polymers can also be used for pH sensing. Conducting

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polymers, e.g., polyaniline, substituted polyaniline, and polythiophene, vary in color as a function of pH. A thin coating of a conducting polymer on the end of the fiber surface can be used to detect the pH of a solution. A fiber-optic pH sensor has been developed based on absorbance changes of the polymers both in the visible and near-IR regions. These pH sensors have the advantage that the polymers can be used over a wide pH range since they are essentially electrolytes with multiple pKa values. These polymers have certain disadvantages such as interference from other ions and the need for reconditioning (with HCl) before each measurement, which is necessary to nullify the conformational changes occurring in the polymer on pH changes. Fiber-optic chemical sensors for different analytes can be developed based on pH sensors. Gases such as CO2 and NH3 react with water and change the pH of a solution when solvated, which can be detected by a pH sensor. Gas fiber-optic chemical sensors will be discussed in the next section. Any chemical or biological species that produces either acids or bases during a chemical or enzymatic reaction can be detected by measuring the pH of the medium. b. Gas and vapor sensing. Gas sensing fiber-optic chemical sensors are mainly based on fluorescence quenching and acid–base chemistry. Optical oxygen sensors are primarily designed based on quenching of a luminescent dye. Fiber-optic chemical sensors for oxygen are constructed by immobilizing oxygen-sensitive fluorophores, using entrapment or adsorption on the fiber surface since most of these dyes do not have an appropriate functional group suitable for covalent immobilization. The process of dynamic quenching is fully reversible, i.e., the dye is not consumed by reaction with oxygen. Dynamic quenching of the fluorophore is responsible for the decrease in fluorescence, and the Stern–Volmer quenching model can be used to express the extent of oxygen quenching: I0 /I = 1 + K SV [O2 ],

(12)

where I is the fluorescence intensity at a particular oxygen concentration, I0 is the value in the absence of oxygen, K SV is the Stern–Volmer quenching constant, and [O2 ] is the concentration of oxygen. A linear calibration is thereby obtained by plotting the intensity ratio as a function of the oxygen concentration. At higher oxygen concentrations, this plot deviates from linearity. Any dye whose fluorescence intensity is quenched by oxygen can be used to construct a fiber-optic oxygen sensor. Commonly used dyes include polyaromatic hydrocarbons (PAH) such as pyrene, fluoranthene, or benzoperylene, and organometallic complexes of ruthenium, osmium, palladium, and platinum. Typical polymeric sup-

ports used for immobilizing these indicators are silicone rubber, plasticized PVC, polystyrene, poly(hydroxyethyl methacrylate) (pHEMA), or porous glass. The sensitivity of the sensor depends on the dye interaction with oxygen and the gas permeability of the polymer film. Sensor selectivity is provided both by the molecular specificity of the quenching phenomenon and the molecular restrictions due to selective permeability of the polymer membrane. Oxygen affects not only the intensity of the indicator fluorescence, but also its decay time. Organometallic dyes have longer fluorescence decay times and can be used to prepare oxygen sensors based on the measurement of this decay time. In comparison to fluorescence-intensity-based sensor types, decay-time-based sensors have less signal drift due to leaching or bleaching because the decay time is not dependent on fluorophore concentration. Fiber-optic chemical sensors for acidic or basic gases such as ammonia, carbon dioxide, hydrogen cyanide, and nitrogen oxide can be constructed by coupling simple acid–base chemistry with pH sensors. First, pH-sensitive dyes or indicators are immobilized using polymers or sol– gel glasses and subsequently covered by a gas-permeable membrane on the distal end of a fiber. The gas-permeable membrane separates the sample solution from the immobilized dye. The acidic or basic gases cross the membrane, enter into the indicator layer, and undergo proton transfer with the dye. The extent of this reaction is monitored spectroscopically through the fiber. For example, a fiber-optic ammonia sensor is prepared based on Dy − H + NH3 → Dy− + NH+ 4.

(13)

Usually the nonprotonated form of the indicator dye is detected either by an absorbance or fluorescence measurement. As the ammonia concentration in the sample increases, the concentration of the nonprotonated dye increases, which causes an increase in the measured fluorescence or absorbance. The measured absorbance A is related to the ammonia concentration in the sample solution by the following expression: A=

εbK eq CDy [NH3 ]s , CNH3 + K eq [NH3 ]s

(14)

where ε is the molar absorptivity of the chromophore, b is the effective path length at the sensor end, K eq is the equilibrium constant of the above reaction, CDy and CNH3 correspond to total dye and total ammonia concentration ([NH+ 4 ] + [NH3 ]) in the dye solution, respectively, and [NH3 ]s is the ammonia concentration in the sample solution. Two different methods can be used to prepare a fiberoptic carbon dioxide sensor. In the first method, the

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sity of the immobilized dye. The concentration of CO2 is determined by measuring the pH of the buffer solution. The second type of CO2 fiber-optic chemical sensor is constructed by using ion pairs consisting of a pH indicator anion and an organic quaternary cation. First, a pH indicator dye (DH) and a quaternary ammonium hydroxide (Q+ OH− ) are entrapped into a proton-impermeable but CO2 -permeable polymer membrane, which is then immobilized onto the fiber’s surface. The mechanism of this CO2 sensor is based on the interaction between the dye molecules (DH) and the quaternary cations (Q+ OH− ) to form hydrated ion pairs (Q+ D− · xH2 O). The hydrated ion pair is dissolved in the polymer, where it reacts with CO2 according to the following reaction: {Q+ D− · xH2 O} + CO2 ⇐⇒ {Q+ HCO− 3 · (x − 1)H2 O} FIGURE 12 Fiber-optic sensor for carbon dioxide with a buffer solution entrapped in a polymer gel at the fiber tip and covered with a CO2 -permeable membrane.

sensors are obtained by entrapping a bicarbonate buffer along with an absorbance or fluorescence indicator into a polymer gel matrix and subsequently covering the matrix by a CO2 -permeable membrane at the fiber tip. A schematic arrangement of such CO2 sensors is shown in Fig. 12. The sensor measures the pH of the bicarbonate buffer solution, which is in equilibrium with CO2 outside the membrane, i.e., membrane

CO2 ←→ CO2 + H2 O, CO2 + H2 O ⇐⇒ H2 CO3 , H2 CO3 ⇐⇒ H+ + HCO− 3,

(15)

HCO− ⇐⇒ H+ + CO2− 3 3 . These reactions are governed by the following equilibrium constants: K 1 = [H+ ][HCO− 3 ]/[CO2 ],  2−  + K 2 = [H ] CO3 [HCO− 3 ].

(16) (17)

By considering the above two equilibria, the equation used to relate the external [CO2 ] to the internal [H+ ] is [H+ ]3 + N [H+ ]2 − (K 1 [CO2 ] + K W )[H+ ] − 2K 1 K 2 [CO2 ] = 0,

(18)

where N is the bicarbonate ion concentration in the internal solution, [CO2 ] is the total analytical concentration of carbon dioxide, i.e., [CO2 ] = [CO2 ](aq) + [H2 CO3 ], and K W = [H+ ][OH− ]. As carbon dioxide crosses the membrane, the microenvironmental pH of the entrapped buffer changes, changing the absorbance or fluorescence inten-

+ DH.

(19)

The indicator dye becomes protonated and changes its absorption (or emission) maximum. Such sensors can be used for determining CO2 in dry gases as well as in aqueous solutions. Fiber-optic chemical sensors for the detecting organic vapors (such as methanol, chloroform, benzene, toluene, and nitroaromatics) are constructed by immobilizing a solvatochromic fluorescent dye into various polymers on the fiber tip. Solvatochromic indicators change their fluorescent properties (such as intensity, emission wavelength maximum) as the polarity of the medium changes. For example, the solvatochromic indicator Nile red exhibits a large shift in emission wavelength maximum with changes in local polarity. Typically, its absorption and/or emission spectra shift to higher wavelengths when exposed to solvents with increasing polarity. A single solvatochromic dye can be immobilized within a polymer matrix, having its own baseline polarity. The sorption of organic vapors into the polymer changes the microenvironmental polarity of the dye, which changes its emission/absorption maximum. Different organic vapors change the polarity, hydrophobicity, and swelling tendency of the polymer, generating different fluorescence responses of the immobilized dye. c. Ion sensing. Several different schemes can be applied to fiber-optic chemical sensors for detecting ions other than hydrogen. One approach is to design a system, similar to pH fiber-optic chemical sensors, in which a dye that selectively binds a metal ion of interest is immobilized in an ion-permeable polymer such as cellulose or a hydrogel at the tip of an optical fiber. The reaction between the dye and the ion changes the absorbance or fluorescence of such dyes. Absorbance or fluorescence intensity changes are measured as a function of ion concentration, but this

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FIGURE 13 The chemical structure of two dyes that possess metal-binding crown ethers. The effects of metal ion chelation will have different photophysical consequences depending on the location of the interaction. This interaction will cause the dyes to display unique emission spectra upon metal binding.

approach is not as selective as other methods (discussed below). The second ion-sensing approach involves the use of fluorogenic and chromogenic crown ethers attached to the fiber tip directly or via an ion-permeable membrane. Fluorogenic and chromogenic crown ethers are prepared by covalently attaching a fluorogenic or a chromogenic dye to crown ethers (some examples are given in Fig. 13). The molecular design of these crown ethers must assure that the spectroscopic properties of the attached dye change when the crown ethers bind to metal ions. Ion-sensitive fiber-optic chemical sensors prepared with crown ethers are highly selective due to chemical recognition for specific metal ions. The sensitivity of this type of fiber-optic chemical sensor for detecting ions in an aqueous environment is relatively low since the formation constants for metal ions binding with the crown ether in water are much lower than in nonaqueous environments. There are two other commonly used schemes for preparing ion-sensitive fiber-optic chemical sensors. The first scheme is based on an ion-exchange technique. A complexing reagent (a cation-selective neutral ionophore) along with a spectroscopically detectable coreagent (fluorescent or absorbant anionic dye) is immobilized inside a thin hydrophobic membrane attached to the fiber. The operating mechanism of this sensor is based on electroneutrality. When analyte ions enter the membrane and selectively bind with the ionophore, an equal number of protons must be released from the membrane (see Fig. 14a). The indicator dye within the membrane acts as the proton donor, thereby altering the measured absorbance or fluorescence. The optical properties of the dye are thereby modulated by the extent of binding of the analyte cation. A constant pH level is maintained by using appropriate buffers. An alternative ion-sensing scheme is referred to as coextraction. In this technique, a highly lipophilic anion such as chloride or salicylate is extracted into the membrane along with a cation, which is usually a proton to maintain electroneutrality. The highly lipophilic nature of the

FIGURE 14 (a) Schematic of the mechanism of proton exchange for a metal ion inside a thin polymer membrane containing an ionophore and a protonated dye. (b) Schematic of the coextrac− − tion procedure. X− 1 is more lipophilic than X2 , hence X1 is more extractable.

anions makes them suitable for extraction by the lipid membrane. Indicator dyes present inside the membrane become protonated and, as a result, the optical properties of the dye change. These changes are easily correlated to the anion concentrations in the aqueous phase. A schematic representation of this sensing scheme is shown in Fig. 14b. Both of these approaches (ion-exchange and coextraction), however, are strongly dependent on pH, which makes them hard to apply to samples where the pH is undefined.

2. Immobilization Techniques Immobilization of sensing materials such as indicators or dyes is a key step in fiber-optic chemical sensor development. The sensing materials employed will largely determine the sensor characteristics for a particular application. Reagent immobilization procedures may involve several steps. The number of immobilization steps should be minimized to maximize yield. A good immobilization method should be (a) simple, (b) fast, (c) general, i.e., the immobilization method can be employed for a variety of sensing materials, (d) stable, i.e., the reagents do not leach from the substrate, and (e) gentle so as to retain the chemical and/or biochemical activities of the species being immobilized. There are three main methods for immobilizing an indicator: adsorption/electrostatic, entrapment, and covalent binding. A schematic representation of these methods is shown in Fig. 15.

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FIGURE 15 Schematic diagram of three different immobilization techniques. (a) Absorption/electrostatic, (b) entrapment, (c) covalent immobilization.

a. Adsorption/electrostatic immobilization. Adsorption immobilization methods involve adsorbing the sensing material onto a solid surface or polymer matrix. Sensing materials can be adsorbed onto the end of optical fibers either directly or with the aid of a thin polymer film. This immobilization method is the simplest of all the methods (described below); however, the adsorbed sensing material tends gradually to leach out from the solid support, decreasing sensing performance and/or lifetime. The surface may first be modified with complementary functional groups to enhance retention of the material. For example, a hydrophobic surface can be prepared to immobilize a hydrophobic species. Electrostatic immobilization employs similar complementarity. The fiber surface can be coated with a thin polyelectrolyte membrane, which interacts electrostatically with oppositely charged sensing materials. For example, a negative surface, such as sulfonated polystyrene, can be prepared to efficiently immobilize a positively charged dye. Similarly, cationic polymers can bind various anions. Ionic indicators or biological molecules (i.e., either cations or anions) can be immobilized by electrostatic interaction.

In order to prevent leaching, strong ion exchangers are employed. The absorption immobilization method is very easy and highly reproducible. Sensing material loading can be controlled easily by changing the immobilization time. Also, the immobilized reagents are easily accessible to analyte because they are situated at sites on the polymer surface. b. Entrapment immobilization. In the entrapment method, the sensing material is physically trapped within a porous matrix. Many different indicators, such as fluorophores or biological materials, may be immobilized in this fashion. When this immobilization method is designed properly, trapped reagents either do not leach out or leach out very slowly from the matrix. The rate of leaching depends on the indicator size, molecular weight, and porosity of the matrix. Organic polymer matrices have been widely used for entrapping sensing materials in the void volume of polymers. Sensing materials are first dissolved in a common solvent with an appropriate polymer or polymer precursor and then the optical fiber is either spin-coated or dip-coated with the viscous solution. Upon

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820 curing or cross-linking, the polymer forms and entraps the material within its pores. Alternatively, complementary solubility between polymer and sensing material can be used to effect the entrapment. For example, lipophilic indicators will dissolve in lipophilic polymers and are not easily leached. Another way to entrap an indicator is to initiate polymerization of a monomer solution containing the indicator. When the polymer is formed, it entraps the indicator. Such polymers can be either thermally or photochemically initiated and attached to the fiber surface by dip-coating procedures. Silanization of the fiber surface with polymerizable groups, such as acrylates or vinyl residues, enhances adhesion of the polymer to the surface. Optically transparent sol–gel glasses are also used for sensing material entrapment. Sol–gel glasses are produced by hydrolysis and polycondensation of organometallic compounds, such as tetraethyl orthosilicate, Si(OCH3 )4 . A sensing material is added to the reaction mixture at some time during the formation of the sol or gel. This viscous sol–gel solution is coated onto an optical fiber to form a sensing element. Sol–gel glasses prepared by this method contain interconnected pores formed by a threedimensional SiO2 network. As a result, the sensing material is trapped but small analytes can readily diffuse in and out of the pores. One advantage of sol–gel glass immobilization is its compatibility with many inorganic and organic reagents allowing many types of sensing materials to be entrapped. Also, sol–gel glasses are chemically, photochemically, and mechanically stable and solvent resistant, and can therefore be useful in harsh conditions. Disadvantages of sol–gel glass immobilization are the slow response times in aqueous media and the fragility of the thin sol–gel glass films compared with polymer films. c. Covalent immobilization. Robust sensing materials are formed by covalent binding to the substrate. Both the substrate and the sensing materials must contain reactive groups for covalent attachment to occur. There are many methods for covalently immobilizing a sensing material to a fiber surface. One simple way is to covalently modify the fiber surface by silanizing with trialkoxysilyl compounds of the type (RO)3 SiR , with R being ethyl or methyl and R being aminopropyl, 3-chloropropyl, 3glycidyloxy, vinyl, or a long-chain amine. The functional group on the fiber surface then reacts with the sensing materials. In some cases, the sensing material must first be activated for reaction with the substrate. For example, dyes possessing COOH groups can be converted to N hydroxysuccinimidoyl esters that can react with an aminemodified surface. Amine-modified surfaces can also be derivatized with amine-containing dyes by using bifunctional cross-linkers such as glutaraldehyde or by direct

Fiber-Optic Chemical Sensors

reaction with an isothiocyanate-containing dye. Sensing materials are also covalently immobilized to a fiber surface by using similar chemistries to those employed with thin polymer or sol–gel glass films. An alternative method for dye immobilization is based on photopolymerization. Dye-doped monomers or dyes containing polymerizable groups can be copolymerized with a monomer on a silanized fiber tip. In these procedures, it is important not to modify the sensing material in such a way as to disturb its ability to bind to the analyte or transduce the signal. Covalent immobilization methods are usually complicated and time-consuming compared with the other two immobilization techniques, but are very reliable since the dye or indicator is not likely to leach out. Covalent binding may result in a reduced response of the sensing material due to bond formation and reduced degrees of freedom upon immobilization. 3. Biological Sensing Materials (Fiber-Optic Based Biosensors) Fiber-optic biosensors are a subtype of fiber-optic chemical sensors that rely on sensing materials of biological origin. In fiber-optic biosensors, biological recognition components such as enzymes, antibodies, DNA, or cells are attached to the optical fiber sensing layer in order to alter the specificity of the sensor. In nature, interaction between biological molecules, such as receptor–ligand, antibody–antigen, or two complementary DNA strands, are highly specific. Some of these recognition molecules can be purified and used in fiberoptic biosensors. Moreover, by using genetic engineering, recombinant recognition molecules can be produced and used. Fiber-optic biosensors can be miniaturized and used in portable analytical devices for clinical, environmental, and industrial applications. Clinical fiber-optic biosensors have been developed for detecting cancer cells, pathogenic bacteria, viruses, toxic proteins, hormones, and drugs. Environmentally important analytes such as pesticides, heavy metals, and carcinogenic compounds can also be detected using fiber-optic biosensors. Industrial applications of fiber-optic biosensors include on-line process monitoring of bacteria or mammalian cell-based bioprocesses. Biological sensing molecules enable the detection of an expanded number of analytes. Such molecules tend to be sensitive to pH or temperature changes and have poor stability, resulting in short lifetimes. Another important limitation is the high cost of purified biological sensing compounds. Biological sensing compounds can be divided into two categories based on their bioactivity: biocatalysts (enzymes and cells) and bioaffinity molecules (antibodies, receptors, and nucleic acids).

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a. Biocatalysts as the recognition element in fiberoptic biosensor. Enzymes are the most commonly employed biocatalysts for fiber-optic biosensor fabrication. These proteins selectively catalyze the conversion of a substrate to product. In fiber-optic biosensors, enzymes are used to catalyze the conversion of a non-optically detectable analyte (the analyte is the enzyme’s substrate) into an optically detectable product. The optical signal obtained, for example, absorbance or fluorescence, is proportional to the product concentration and thereby to the analyte’s concentration. The enzymatic reaction products are measured either directly, if they are optically detectable, or indirectly by using indicators as described in Section III.B.1. Indicators are used to measure common reaction products such as H+ , ammonia, oxygen, carbon dioxide, and hydrogen peroxide. An example of this approach is the glucose biosensor. The enzyme glucose oxidase catalyzes the oxidation of glucose with oxygen (the substrates) to produce gluconolactone and H2 O2 . The glucose concentration is determined by using an indicator to measure either the amount of oxygen consumed or the amount of H2 O2 produced using an appropriate indicator,

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821 bioaffinity fiber-optic biosensors are based on transducing antibody–antigen (analyte) interactions into an optical signal that is proportional to the antigen concentration. Several detection schemes are employed; the simplest one involves the detection of intrinsically fluorescent analytes such as polynuclear aromatic hydrocarbons (PAHs). Antibodies are immobilized on the fiber surface and a fluorescence signal is obtained when the analyte (antigen) is present, as shown in Fig. 16a. A more generalized detection scheme, often called a competition assay, is based on competition for the antibody-binding site between an externally added fluorescent-labeled antigen and the antigen present in the sample (analyte), as shown in Fig. 16b. The antibody is immobilized on the optical fiber surface, a known concentration of fluorescent-labeled antigen is added, and the fluorescence signal obtained is set as the initial signal. To perform an analysis, the same fluorescent-labeled antigen concentration is mixed with a sample containing an unknown antigen concentration.

glucose oxidase

Glucose + O2 −−−−−−−−→
Gluconic acid + H2 O2 . The use of enzymes as sensing materials for fiber-optic biosensors is relatively limited since the stability and the activity of most enzymes significantly decrease when they are removed from their natural environment inside the cells, although immobilization techniques may help. To overcome this stability problem, whole cells may be used as biocatalysts. Although some of the fiber-optic biosensor specificity is reduced with whole-cells, the enzymes within the cells function more efficiently because they are in an optimum environment containing all the necessary cofactors. Whole-cell biocatalysts are particularly advantageous when the detection is based on a sequence of multiple enzymatic reactions. These enzymatic cascade reactions are very difficult and complicated to accomplish by coimmobilizing the enzymes but are relatively straightforward by employing whole cells. The methods for detecting the products in cell-based fiber-optic biosensors are similar to those employed in enzyme fiber-optic biosensors. b. Bioaffinity as the recognition mechanism in fiber-optic biosensors. Antibodies, receptors, and nucleic acids are highly selective and can recognize their binding partners. When the affinity binding reaction is in equilibrium and readily reversible, a sensor results. If the reaction is essentially irreversible, the resulting sensor is called a probe, as it must be regenerated or recharged before it can be used to make another measurement. Most

FIGURE 16 Schematic principles of bioaffinity fiber-optic biosensors. (a) Detection of intrinsically fluorescent molecule using immobilized antibody. (b) Competition assay using a fluorescentlabeled antigen. (c) Sandwich immunoassay using an immobilized antibody and a fluorescent-labeled antibody.

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FIGURE 17 Principle of DNA fiber-optic biosensors. (a) Single-strand DNA probe molecules, with a sequence complementary to one strand of the target DNA sequence, are immobilized onto the fiber. (b) The fluorescent-labeled sample DNA molecules are first dehybridized and the fiber is dipped into the sample solution. (c) After hybridization, the complementary strands of the target DNA are attached to the probe DNA on the fiber and a fluorescence signal is obtained.

When this mixture is analyzed using the fiber-optic biosensor, the resulting fluorescence signal obtained is lower than the initial signal. The relative decrease in the initial signal is proportional to the analyte concentration in the sample. A sandwich immunoassay is another widely used detection scheme and involves the use of two antibodies. The first antibody is immobilized on the fiber and is used to capture the antigen, and the second antibody, which is conjugated to a fluorescent dye or enzyme, is used to generate the signal (Fig. 16c). When an enzyme is used for antibody labeling, the enzymatic conversion of a nonfluorescent substrate to a fluorescent product is measured. The enzyme-labeling method is more sensitive since the signal is amplified by the enzymatic reaction.

Nucleic acid base pairing can also be used for bioaffinity sensor fabrication. The presence of a specific DNA sequence, the “target,” among millions of other different sequences is detected by hybridization to its complementary DNA sequence, the “probe,” which is immobilized on the optical fiber, as shown in Fig. 17. The sample DNA is labeled using fluorescent primers and the polymerase chain reaction (PCR). The resulting double-strand DNA molecules are dehybridized (usually by heating) and then allowed to rehybridize (by cooling) to the single-strand DNA probe molecules immobilized on the fiber surface. If the complementary target DNA sequence is present in the sample, a fluorescence signal is detected on the sensor. The target sequence can be, for example, a unique sequence found only in specific pathogenic bacteria. DNA

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can be easily extracted from water, wastewater, or clinical samples and the presence of pathogenic microorganisms can be determined by the sensor. In general, such DNAsensing schemes are rarely conducted using a single probe sequence but are usually performed with an array of hundreds to thousands of probes.

IV. APPLICATIONS OF FIBER-OPTIC CHEMICAL SENSORS A. Clinical Applications Clinical analytical devices are important for various diagnostics applications since the use of these devices can directly lead to more efficient and effective medical treatment. Most diagnostic tests are performed in a centralized laboratory. Samples must be collected with the attendant transport, storage, and chain-of-custody issues. The remote location of the laboratory delays the medical diagnosis. For these reasons, analytical devices such as sensors that can be located near the patient’s bed or even inside the patient’s body are of great utility. In recent years, fiberoptic chemical sensors have been developed for both in vivo diagnostics (monitoring physical and chemical parameters inside the body) and for clinical sample analysis at the patient bedside (i.e., blood or urine tests). The principal advantage of fiber-optic chemical sensors over electrochemical technologies is that no electrical current is involved in the measurement. Thus, the measurement inside the body is much safer, and analysis at the patient’s bedside does not affect other electrical medical devices. 1. Fiber-Optic Chemical Sensors for In Vivo Analysis In vivo analytical devices ideally should be capable of monitoring several different physiological parameters simultaneously without interfering with an ongoing medical procedure, such as surgery. The devices should be biocompatible, simple to implement and operate, and highly reliable and safe. Fiber-optic chemical sensors can meet most of these requirements since the optical fibers are small (few hundred micrometers in diameter), flexible, nontoxic, and chemically inert. Optical fibers have already proven to be valuable for in vivo clinical applications such as endoscopic procedures and laser power transmission for surgical applications. Typical fiber-optic chemical sensors for in vivo monitoring are constructed from optical fibers that are connected to an external compact unit containing the light source and the detector, as shown in Fig. 18. The optical fiber’s distal end is inserted into the patient through a catheter.

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823 Fiber-optic chemical sensors were originally used to measure oxygen saturation in the blood. The oxygen carrier in the blood is hemoglobin (Hb) and the blood saturation is the ratio (as a percentage) between the oxygen that is actually carried by Hb and the Hb’s maximum carrying capacity. The measurement is based on the large difference in the light absorption of Hb and OxyHb (oxyhemoglobin, i.e., oxygen bound to Hb) at λ = 660 nm. Typically the light is delivered through two bifurcated fibers at two different wavelengths, 660 nm (where Hb absorption is higher than that of OxyHb) and 805 nm (where there is a small difference in absorption between Hb and OxyHb); the absorption is measured and the blood saturation is calculated. Fiber-optic chemical sensors have been developed for measuring all three blood gas parameters pH, PO2 , and PCO2 . These parameters are used to determine the efficacy of gas exchange within tissue and are crucial in surgical procedures such as heart bypass surgery and critical care monitoring for patients with compromised respiratory conditions. The sensing mechanism is based on dyes that change their fluorescence intensities as a function of analyte concentration (see Section III.B.1). Several sensor configurations have been developed for intravascular measurement including one that incorporates the three different sensors into a single device. At present these sensors have not been implemented because of blood compatibility problems in which a thrombus (clot) forms around the sensor tip and affects the measurement accuracy. Several pH in vivo fiber-optic chemical sensors for other clinical applications have been developed. These include pH monitoring in the stomach and the detection and examination of malignant tumors. Both sensors are based on pH-sensitive fluorescent dyes. In gastric sensors, the dyes are immobilized on the fiber tip and the emitted light intensities are correlated to pH changes. Since a wide pH range (1–8) is monitored, there is a need to use several different dyes (each for a different pH range). pH sensors have also been used for locating suspicious tissue and determining if malignancy is present. This oncological sensor is based on the observation that malignant tumors can induce a decrease in their microenvironment pH. Thus, by inserting the optical fiber into the tissue and then injecting a nontoxic pH indicator (fluorescein based), it is possible to identify and map malignant tumor locations. An interesting fiber-optic chemical sensor device for in vivo gastric diagnostics is the Bilitec 2000 (Medtronic Synectics AB). This device is used to measure the bilirubin concentration in the stomach and the esophagus. The bilirubin concentrations are related to bile-containing reflux in these organs and can reveal several pathological conditions such as gastric ulcers and gastric cancer. The device, shown in Fig. 18, consists of two light-emitting

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FIGURE 18 Bilitec 2000 fiber-optic chemical sensor for in vivo gastric diagnostics. (a) The external unit and the optical fibers. (b) The optical fiber’s distal end that is inserted into the body. [Reproduced with permission from Medtronic Synectics AB.]

diodes that emit light at λ = 465 nm (bilrubin absorption) and 570 nm (reference). The light is transmitted by the fiber bundles to the probe (miniaturized spectrophotometric cell) at the fiber’s distal end. Bilirubin’s light absorption is measured by the detector and bilirubin concentrations are calculated. Several clinical fiber-optic chemical sensors are commercially available and others are in different stages of development. The demand for in vivo fiber-optic chemical sensors for monitoring important analytes such as glucose, potassium, urea, lactate, and some enzymes has led to concerted research efforts in this field. 2. Clinical Fiber-Optic Chemical Sensors for In Situ Sample Analysis Fiber-optic chemical sensors can be located at the patient’s bedside or even in the patient’s home for self-use. Such sensors are designed as compact, simple–to-use devices. While these sensors employ essentially the same sensing chemistry as with in vivo sensors, the overall sensor design can be much simpler and a wider range of materials may be used for sensor fabrication. Fiber-optic chemical sensors for blood gases (PO2 , PCO2 , and pH) are commercially available. Measurements are based on the use of immobilized fluorescent dyes. In one device, the dyes are incorporated into a disposable apparatus that is inserted into an extracorporeal blood circuit on one side and connected to a fiber bundle on the other. These sensors are mainly used to monitor blood gases during open heart surgery. Another approach is to measure pH and blood gases using a paracorporeal measurement at the patient’s bedside. The sensors are three fluorescent-sensing materials responsive to each of the blood gases. The sensors are placed into an external tube connected to an arterial blood line. Blood samples are periodically and automatically pumped into the tube, analyzed by the sensors, and then returned to the blood line. In this way, the blood can be monitored

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semicontinuously without requiring blood samples to be taken from the patient. Fiber-optic biosensor can be potentially used for clinical sample analysis. As described above, by using enzymes and antibodies immobilized on the fiber tip or around the fiber core (evanescent field), different fiber-optic biosensors have been developed. In most cases, the measurement is based on changes in fluorescence intensity. The target analytes include glucose, cholesterol, enzymes, antibodies, bacteria, and viruses. Measurements are usually fast and simple, and in many devices, the probe is disposable; however, the instability of the biological recognition materials reduces the sensor lifetime. Recently, fiber-optic biosensors for detecting DNA sequences have been reported. These fiber-optic biosensors can be used for detecting pathogenic microorganisms and for identifying defective genes. The sensors are usually designed in an array format (Fig. 19) allowing the simultaneous analysis of hundreds or thousands of samples. Although just recently developed, these sensors will undoubtedly revolutionize the clinical diagnostics field. B. Environmental Fiber-Optic Chemical Sensors Both short- and long-term harmful effects of toxic chemical accumulation in the environment have become apparent in the last few decades. In order to reduce the environmental damage caused by these pollutants, regulations were set that either restricted or completely prohibited the release of environmentally hazardous chemicals into the environment. The efficient enforcement of these regulations is highly dependent on sensitive and reliable analytical tools for environmental monitoring. These analytical tools ideally should be sensitive, simple to construct, portable, and suitable for continuous field (i.e., in situ) measurements. Fiber-optic chemical sensor systems can meet most of these requirements since they can be miniaturized and integrated into portable analytical devices that can offer high specificity and sensitivity, fast response, mechanical stability, and low cost. Furthermore, by using long optical fibers, the sensors can be used for remote analytical monitoring. Most commercially available environmental fiber-optic chemical sensors are for pH and oxygen monitoring in water and wastewater. The monitoring of unusual changes in oxygen and pH can be used as an indirect indication of the presence of pollutants. These sensors are based on fluorescent dyes as the sensing material. The optical fiber and the sensing elements are covered with a metal jacket that provides immunity from harsh environmental conditions. Environmental sensors capable of detecting specific pollutants such as volatile organic compounds and heavy metals (two of the largest classes of chemical pollutants)

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FIGURE 19 Fluorescence image of a DNA sensor array with ∼13,000 DNA probe regions.

have also been developed. For detecting volatile organic compounds, direct fluorescence spectroscopy can be used since most volatile organic compounds have unique fluorescence spectra. Moreover, by using time-resolved fluorescence spectroscopy, it is possible to distinguish between volatile organics with similar structures, for example, toluene and benzene. Heavy-metal fiber-optic chemical sensors are mostly based on ion sensitive indicators as described in Section III.B.1.c. Pesticides are another important group of pollutants that can be detected by fiber-optic chemical sensors. Since pesticides are designed to interact with biological molecules, fiber-optic biosensors are mostly used for their detection. One example is the detection of organophosphate and carbamate pesticides by monitoring their inhibition effect on the enzymatic reaction of acetylcholinesterase (AChE) with its substrate, acetylcholine. The enzyme is coimmobilized at the distal end of the fiber together with

a pH-sensitive dye. The formation of acetic acid, which is one of the enzymatic reaction products, changes the local pH and thereby the fluorescence signal. The inhibition of this reaction can be related to the pesticide concentration in the sample. Current research activities on environmental fiber-optic chemical sensors are focused on developing multianalyte sensors for on-line and in situ monitoring. These sensors may one day be used as environmental warning devices that will help prevent environmental catastrophes by triggering an alarm whenever uncontrolled release of pollutants to the environment occurs. C. Industrial and Bioprocess Control Applications In the chemical and bioprocess industries, the need for real-time process and quality control has elicited a great

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826 interest in chemical sensors. Effective process control is a basic requirement for the optimal utilization of chemical and biological materials. Conventional analytical techniques for measuring industrial or biological materials such as GC, HPLC, and flow injection analysis have several drawbacks such as price, large instrument size, interference by medium components, and drift. Intrinsic fiber-optic chemical sensors are widely used in industry and bioprocess control. Two basic approaches are used in remote applications. In the first approach, the spectroscopic parameters are chosen such that the analyte gives a unique signal compared with all other components in the sample. Examples of such approaches are as follows: (a) Fiber-optic remote fluorescence spectroscopy is used to measure reduced nicotinamide adenine dinucleotide (NADH) in bioreactors. The idea is to correlate the viability of a population of cells within the reactor to the total amount of NADH